Human Cholangiocarcinoma 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.

A metabolomics approach reveals metabolic disturbance of human cholangiocarcinoma cells after parthenolide treatment

Ethnopharmacological relevanceTanacetum parthenium (L.) Schultz-Bip, commonly known as feverfew, has been traditionally used to treat fever, migraines, rheumatoid arthritis, and cancer. Parthenolide (PTL), the main bioactive ingredient isolated from the shoots of feverfew, is a sesquiterpene lactone with anti-inflammatory and antitumor properties. Previous studies showed that PTL exerts anticancer activity in various cancers, including hepatoma, cholangiocarcinoma, acute myeloid leukemia, breast, prostate, and colorectal cancer. However, the metabolic mechanism underlying the anticancer effect of PTL remains poorly understood. Aim of the studyTo explore the anticancer activity and underlying mechanism of PTL in human cholangiocarcinoma cells. Material and methodsIn this investigation, the effects and mechanisms of PTL on human cholangiocarcinoma cells were investigated via a liquid chromatography/mass spectrometry (LC/MS)-based metabolomics approach. First, cell proliferation and apoptosis were evaluated using cell counting kit-8 (CCK-8), flow cytometry analysis, and western blotting. Then, LC/MS-based metabolic profiling along with orthogonal partial least-squares discriminant analysis (OPLS-DA) has been constructed to distinguish the metabolic changes between the negative control group and the PTL-treated group in TFK1 cells. Next, enzyme-linked immunosorbent assay (ELISA) was applied to investigate the changes of metabolic enzymes associated with significantly alerted metabolites. Finally, the metabolic network related to key metabolic enzymes, metabolites, and metabolic pathways was established using MetaboAnalyst 5.0 and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Database. ResultsPTL treatment could induce the proliferation inhibition and apoptosis of TFK1 in a concentration-dependent manner. Forty-three potential biomarkers associated with the antitumor effect of PTL were identified, which primarily related to glutamine and glutamate metabolism, alanine, aspartate and glutamate metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, arginine biosynthesis, arginine and proline metabolism, glutathione metabolism, nicotinate and nicotinamide metabolism, pyrimidine metabolism, fatty acid metabolism, phospholipid catabolism, and sphingolipid metabolism. Pathway analysis of upstream and downstream metabolites, we found three key metabolic enzymes, including glutaminase (GLS), γ-glutamyl transpeptidase (GGT), and carnitine palmitoyltransferase 1 (CPT1), which mainly involved in glutamine and glutamate metabolism, glutathione metabolism, and fatty acid metabolism. The changes of metabolic enzymes associated with significantly alerted metabolites were consistent with the levels of metabolites, and the metabolic network related to key metabolic enzymes, metabolites, and metabolic pathways was established. PTL may exert its antitumor effect against cholangiocarcinoma by disturbing metabolic pathways. Furthermore, we selected two positive control agents that are considered as first-line chemotherapy standards in cholangiocarcinoma therapy to verify the reliability and accuracy of our metabolomic study on PTL. ConclusionThis research enhanced our comprehension of the metabolic profiling and mechanism of PTL treatment on cholangiocarcinoma cells, which provided some references for further research into the anti-cancer mechanisms of other drugs.

Cisplatin Disrupts Proteasome Bounce-Back Effect through Suppressing ZEB1/Nfe2l1 in Cholangiocarcinoma.

Bortezomib (BTZ) is a powerful proteasome inhibitor that has been approved for the treatment of haematologic malignancies. Its effectiveness has been assessed against different types of solid tumours. BTZ is ineffective in most solid tumours because of drug resistance, including cholangiocarcinoma, which is associated with a proteasome bounce-back effect. However, the mechanism through which proteasome inhibitors induce the proteasome bounce-back effect remains largely unknown. Cholangiocarcinoma cells were treated with BTZ, cisplatin, or a combination of both. The mRNA levels of Nfe2l1 and proteasome subunit genes (PSMA1, PSMB7, PSMD1, PSMD11, PSMD14, and PSME4) were determined using quantitative real time polymerase chain reaction (qPCR). The protein levels of nuclear factor-erythroid 2-related factor 1 (Nfe2l1) and proteasome enzyme activity were evaluated using western blotting and proteasome activity assays, respectively. Transcriptome sequencing was performed to screen for potential transcription factors that regulate Nfe2l1 expression. The effect of zinc finger E-box-binding homeobox 1 (ZEB1) on the expression of Nfe2l1 and proteasome subunit genes, as well as proteasome enzyme activity, was evaluated after the knockdown of ZEB1 expression with siRNA before treatment with BTZ. The transcriptional activity of ZEB1 on the Nfe2l1 promoter was detected using dual-luciferase reporter gene and chromatin immunoprecipitation assays. Cell viability was measured using the cell counting kit-8 (CCK-8) assay and cell apoptosis was assessed using western blotting and flow cytometry. Cisplatin treatment of BTZ-treated human cholangiocarcinoma cell line (RBE) suppressed proteasome subunit gene expression (proteasome bounce-back) and proteasomal enzyme activity. This effect was achieved by reducing the levels of Nfe2l1 mRNA and protein. Our study utilised transcriptome sequencing to identify ZEB1 as an upstream transcription factor of Nfe2l1, which was confirmed using dual-luciferase reporter gene and chromatin immunoprecipitation assays. Notably, ZEB1 knockdown using siRNA (si-ZEB1) hindered the expression of proteasome subunit genes under both basal and BTZ-induced conditions, leading to the inhibition of proteasomal enzyme activity. Furthermore, the combination treatment with BTZ, cisplatin, and si-ZEB1 significantly reduced the viability of RBE cells. Our study uncovered a novel mechanism through which cisplatin disrupts the BTZ-induced proteasome bounce-back effect by suppressing the ZEB1/Nfe2l1 axis in cholangiocarcinoma. This finding provides a theoretical basis for developing proteasome inhibitor-based strategies for the clinical treatment of cholangiocarcinoma and other tumours.

Ultra-low content physio-chemically crosslinked gelatin hydrogel improves encapsulated 3D cell culture

Gelatin-based hydrogels are extensively used for 3D cell culture, bioprinting, and tissue engineering due to their cell-adhesive nature and tunable physio-chemical properties. Gelatin hydrogels for 3D cell culture are often developed using high-gelatin content (frequently 10–15 % w/v) to ensure fast gelation and improved stability. While highly stable, such matrices restrict the growth of encapsulated cells due to creating a dense, restrictive environment around the encapsulated cells. Hydrogels with lower polymer content are known to improve 3D cell growth, yet fabrication of ultra-low concentration gelatin hydrogels is challenging while ensuring fast gelation and stability. Here, we demonstrate that physical gelation and photo-crosslinking in gelatin results in a fast-gelling hydrogel at a remarkably low gelatin concentration of 1 % w/v (GelPhy/Photo). The GelPhy/Photo hydrogel was highly stable, allowed uniform 3D distribution of cells, and significantly improved the spreading of encapsulated 3T3 fibroblast cells. Moreover, human cholangiocarcinoma (HuCCT-1) cells encapsulated in 1 % GelPhy/Photo matrix grew and self-assembled into epithelial cysts with lumen, which could not be achieved in a traditional high-concentration gelatin hydrogel. These findings pave the way to significantly improve existing gelatin hydrogels for 3D cell culture applications.

The Human Placental Amniotic Membrane Mesenchymal-Stromal-Cell-Derived Conditioned Medium Inhibits Growth and Promotes Apoptosis of Human Cholangiocarcinoma Cells In Vitro and In Vivo by Suppressing IL-6/JAK2/STAT3 Signaling.

Mesenchymal stromal cells (MSCs) have recently been shown to play an important role in the growth and progression of many solid tumors, including cholangiocarcinoma (CCA). The human placental amniotic membrane (hPAM) is one of the most favorable sources of MSCs due to its availability and non-invasive harvesting procedure. However, the role of human placental amniotic membrane mesenchymal stromal cells (hPAMSCs) in the growth and progression of human CCA has not yet been determined. This study investigates the effects of conditioned medium derived from hPAMSCs (PA-CM) on the properties of three human CCA cell lines and explores possible mechanisms of action. Varying concentrations of PA-CM were used to treat CCA cells to determine their effects on the proliferation and apoptosis of CCA cells. The results showed that PA-CM inhibited the proliferation and colony-forming capacity of KKU100, KKU213A, and KKU213B cells. PA-CM also promoted the apoptosis of these CCA cells by causing the loss of mitochondrial membrane potential. Western Blotting confirmed that PA-CM induced CCA cell apoptosis by increasing the levels of the Bax/Bcl-2 ratio, cleaved caspase 3, and cleaved PARP, possibly by inhibiting the IL-6/JAK2/STAT3 signaling pathway. Moreover, our in vivo study also confirmed the suppressive effect of hPAMSCs on CCA cells by showing that PA-CM reduced tumor volume in nude mice transplanted with human CCA cells. Taken together, our results demonstrate that PA-CM has potent tumor-suppressive effects on human CCA cells and could potentially be used in combination with chemotherapy to develop a more effective treatment for CCA patients.

Tetraspanins from the liver fluke Opisthorchis viverrini stimulate cholangiocyte migration and inflammatory cytokine production.

The liver fluke Opisthorchis viverrini (Poirier, 1886) (Digenea) secretes extracellular vesicles (EVs) bearing CD63-like tetraspanins on their surface. Fluke EVs are actively internalised by host cholangiocytes in the bile ducts, where they drive pathology and promote neoplasia through induction of cellular proliferation and secretion of inflammatory cytokines. We investigated the effects of tetraspanins of the CD63 superfamily by co-culturing recombinant forms of the large extracellular loop (LEL) of O. viverrini tetraspanin-2 (rLEL-Ov-TSP-2) and tetraspanin-3 (rLEL-Ov-TSP-3) with non-cancerous human bile duct (H69) and cholangiocarcinoma (CCA, M213) cell lines. The results showed that cell lines co-cultured with excretory/secretory products from adult O. viverrini (Ov-ES) underwent significantly increased cell proliferation at 48 hours but not 24 hours compared to untreated control cells (P < 0.05), whereas rLEL-Ov-TSP-3 co-culture resulted in significantly increased cell proliferation at both 24 hours (P < 0.05) and 48 hours (P < 0.01) time points. In like fashion, H69 cholangiocytes co-cultured with both Ov-ES and rLEL-Ov-TSP-3 underwent significantly elevated Il-6 and Il-8 gene expression for at least one of the time points assessed. Finally, both rLEL-Ov-TSP-2 and rLEL-Ov-TSP-3 significantly enhanced migration of both M213 and H69 cell lines. These findings indicated that O. viverrini CD63 family tetraspanins can promote a cancerous microenvironment by enhancing innate immune responses and migration of biliary epithelial cells.

Protein Modification via Nitrile Oxide-Dehydroalanine Cycloaddition: Formation of Isoxazoline Ring on the Protein Backbone.

Here we describe a novel catalyst-free 1,3-dipolar cycloaddition bioconjugation approach for chemical modification of proteins. The dehydroalanine (Dha)-containing protein reacts with nitrile oxides generated in situ through 1,3-dipolar cycloaddition in fully aqueous-buffered systems. This leads to the formation of a new isoxazoline ring at a pre-defined site (Dha) of the protein. Furthermore, the 1-pyrene isoxazoline-installed annexin V acts as a fluorescent probe, which successfully labels the outer cellular membranes of human cholangiocarcinoma (HuCCA-1) cells for detection of apoptosis.

Radioactive 125I Seed Inhibits Cell Migration and Invasion and Promotes Apoptosis by Inactivating the VEGFR2 Signaling Pathway in Cholangiocarcinoma.

To investigate the potential mechanisms of 125I seed implantation therapeutic treatment on inactivating the VEGFR2/PI3K/AKT pathway in cholangiocarcinoma. The human cholangiocarcinoma cell lines HCCC-9810 and HuCCT1 were purchased for in vitro studies. The BALB/c nude mice were obtained for in vivo studies. The proliferation of cells was detected by CCK-8, colony formation, and BrdU staining. The migration and invasion of cells were determined by wound healing assay and Transwell assay, respectively. Hematoxylin and eosin staining was utilized for histological evaluation. Protein expression was determined by western blotting and immunohistochemistry. Compared with the control group, .6mCi group and .8mCi group inhibited cholangiocarcinoma cells proliferation, invasion, migration, and promoted apoptosis, the protein expression of p-VEGFR2, VEGFR2, PI3K, p-AKT/AKT, cyclin B1, cyclin A, CDK1, and Bcl-2 was decreased. Similar results were obtained from in vitro experiments. However, when VEGF is overexpressed, the inhibitory effect of .8mCi was partially significantly reversed on cholangiocarcinoma cells. The in vivo studies further confirmed the inhibitory effects of .6mCi group and .8mCi group on cholangiocarcinoma. 125I seed irradiation could inhibit cholangiocarcinoma cells proliferation, migration, and invasion and promote apoptosis through inactivation of the VEGFR2/PI3K/AKT signaling pathway.

Lanatoside C decelerates proliferation and induces apoptosis through inhibition of STAT3 and ROS-mediated mitochondrial membrane potential transformation in cholangiocarcinoma.

Introduction: The incidence of cholangiocarcinoma (CCA) has increased worldwide in recent years. Given the poor prognosis associated with the current management approach of CCA, new therapeutic agents are warranted to improve the prognosis of this patient population. Methods: In this study, we extracted five cardiac glycosides (CGs) from natural plants: digoxin, lanatoside A, lanatoside C, lanatoside B, and gitoxin. Follow-up experiments were performed to assess the effect of these five extracts on cholangiocarcinoma cells and compounds with the best efficacy were selected. Lanatoside C (Lan C) was selected as the most potent natural extract for subsequent experiments. We explored the potential mechanism underlying the anticancer activity of Lan C on cholangiocarcinoma cells by flow cytometry, western blot, immunofluorescence, transcriptomics sequencing, network pharmacology and in vivo experiments. Results: We found that Lan C time-dependently inhibited the growth and induced apoptosis of HuCCT-1 and TFK-1 cholangiocarcinoma cells. Besides Lan C increased the reactive oxygen species (ROS) content in cholangiocarcinoma cells, decreased the mitochondrial membrane potential (MMP) and resulted in apoptosis. Besides, Lan C downregulated the protein expression of STAT3, leading to decreased expression of Bcl-2 and Bcl-xl, increased expression of Bax, activation of caspase-3, and initiation of apoptosis. N-acetyl-L-cysteine (NAC) pretreatment reversed the effect of Lan C. In vivo, we found that Lan C inhibited the growth of cholangiocarcinoma xenografts without toxic effects on normal cells. Tumor immunohistochemistry showed that nude mice transplanted with human cholangiocarcinoma cells treated with Lan C exhibited decreased STAT3 expression and increased caspase-9 and caspase-3 expression in tumors, consistent with the in vitro results. Conclusion: In summary, our results substantiates that cardiac glycosides have strong anti-CCA effects. Interestingly the biological activity of Lan C provides a new anticancer candidate for the treatment of cholangiocarcinoma.

Flavokawain B Inhibits Growth of Cholangiocarcinoma Cells by Suppressing the Akt Pathway.

Cholangiocarcinoma is a devastating malignancy with limited treatment options and poor prognosis. Natural products have gained considerable attention for showing antitumor effects with less toxicities. Flavokawain B (FKB), a natural product, has been studied for its antitumor effects on various cancer cells. However, the anti-tumor effect of FKB on cholangiocarcinoma cells remains unknown. This study aimed at investigating the antitumor effect of FKB on cholangiocarcinoma cells in vitro and in vivo. SNU-478, a human cholangiocarcinoma cell line, was used in this study. Effects of FKB on cell growth inhibition and apoptosis were investigated. The synergistic anti-tumor effect of FKB and cisplatin in combination was also evaluated. Western blotting was performed to examine the underlying molecular mechanisms of the effect of FKB. A xenograft mouse model study was performed to investigate the effect of FKB in vivo. FKB inhibited cell proliferation of cholangiocarcinoma cells in a concentration- and time-dependent manner. FKB also induced cellular apoptosis additively in combination with cisplatin. Akt pathway was suppressed by FKB either alone or in combination with cisplatin. In the xenograft model, FKB treatment in combination with cisplatin/gemcitabine significantly inhibited tumor growth of SNU-478 cells. FKB showed an antitumor effect through the induction of apoptosis, which was mediated by suppressing the Akt pathway in cholangiocarcinoma cells. However, the synergistic effect of FKB and cisplatin was not definite.

Establishment and Characterization of a New Human Intrahepatic Cholangiocarcinoma Cell Line LIV27.

Simple SummaryCholangiocarcinoma (CCA) is usually diagnosed at a late stage and has a poor prognosis. Cell lines serve as useful models for testing scientific questions in vitro and in vivo. To aid scientific discovery for the purpose of improved early detection and treatment, we developed a CCA cell line, designated LIV27, from a surgically resected intrahepatic CCA in a Caucasian patient with primary sclerosing cholangitis (PSC). LIV27 has high tumorigenicity in nude mice and expands the availability of intrahepatic CCA cell lines.Cholangiocarcinoma (CCA) is a highly lethal cancer arising from the biliary tract epithelium. The cancer biology of this neoplasm is not well understood. To date, only a few CCA cell lines have been reported, which were mostly developed from Asian patients. In this study, we report and characterize a new intrahepatic CCA cell line, LIV27, derived from a surgically resected tumor in a 67-year-old Caucasian woman with primary sclerosing cholangitis (PSC). LIV27 cells grow well in collagen-coated flasks or plates with a doubling time of 57.8 h at passage 14. LIV27 cells have high tumorigenicity in nude mice and stain positive for CK7 and CK19, markers that differentiate CCA from hepatocellular carcinoma. Karyotype analysis showed that LIV27 is aneuploid. We established a single-locus short tandem repeat profile for the LIV27 cell line. This newly established cell line will be a useful model for studying the molecular pathogenesis of, and developing novel therapies for, cholangiocarcinoma.

Suppression of intrahepatic cholangiocarcinoma cell growth by SKI via upregulation of the CDK inhibitor p21.

Cholangiocarcinoma (CC) has a poor prognosis and different driver genes depending on the site of onset. Intrahepatic CC is the second-most common liver cancer after hepatocellular carcinoma, and novel therapeutic targets are urgently needed. The present study was conducted to identify novel therapeutic targets by exploring differentially regulated genes in human CC. MicroRNA (miRNA) and mRNA microarrays were performed using tissue and serum samples obtained from 24 surgically resected hepatobiliary tumor cases, including 10 CC cases. We conducted principal component analysis to identify differentially expressed miRNA, leading to the identification of miRNA-3648 as a differentially expressed miRNA. We used an in silico screening approach to identify its target mRNA, the tumor suppressor Sloan Kettering Institute (SKI). SKI protein expression was decreased in human CC cells overexpressing miRNA-3648, endogenous SKI protein expression was decreased in human CC tumor tissues, and endogenous SKI mRNA expression was suppressed in human CC cells characterized by rapid growth. SKI-overexpressing OZ cells (human intrahepatic CC cells) showed upregulation of cyclin-dependent kinase inhibitor p21 mRNA and protein expression and suppressed cell proliferation. Nuclear expression of CDT1 (chromatin licensing and DNA replication factor 1), which is required for the G1/S transition, was suppressed in SKI-overexpressing OZ cells. SKI knockdown resulted in the opposite effects. Transgenic p21-luciferase was activated in SKI-overexpressing OZ cells. These data indicate SKI involvement in p21 transcription and that SKI-p21 signaling causes cell cycle arrest in G1, suppressing intrahepatic CC cell growth. Therefore, SKI may be a potential therapeutic target for intrahepatic CC.

Evaluation of In Vitro Cytotoxic Property Against Cholangiocarcinoma Cell Line and GC/MS Analysis from Leaf of Erythrophleum succirubrum Gagnep.

Plants are valuable sources of new pharmaceuticals. Secondary metabolites of the genus Erythrophleum exhibit cytotoxicity and may have therapeutic value. The cytotoxic activity of ethanolic leaf extract of Erythrophleum succirubrum Gagnep. against a human cholangiocarcinoma cell line was assessed. Crude extract of E. succirubrum was prepared by ethanol extraction. The ethanolic leaf extract of E. succirubrum was evaluated for cytotoxicity against the human cholangiocarcinoma cell line KKU-M213 using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assays. The chemical composition of E. succirubrum leaf extract was analyzed using GC/MS. The ethanolic leaf extract of E. succirubrum reduced the viability of KKU-M213 cells in a dose- and time-dependent manner. It showed high cytotoxicity, with IC50 values of 65.22 ± 1.18 µg/mL and 1.19 ± 1.38 µg/mL at exposure times of 24 and 96 h, respectively. GC/MS analysis of the ethanolic leaf extract of E. succirubrum identified 22 components. The main constituents identified were Cyclohexanone, 2-[2-nitro-1-(2-naphthyl)ethyl]-(14.79%) followed by allomycin (14.65%), mome inositol (14.30%), campesterol (11.80%) and ethyl linolenate (10.83%), respectively. Five major groups of compounds were found, with lipids dominating, followed by carbohydrates, benzenoids, phenylpropanoids, polyketides and organoheterocyclic compounds. Many of the bioactive components discovered in the ethanolic leaf extract of E. succirubrum might be responsible for its cytotoxic properties.

Genistein Sensitizes Human Cholangiocarcinoma Cell Lines to Be Susceptible to Natural Killer Cells.

Simple SummaryCholangiocarcinoma (CCA) is a malignancy of bile duct epithelial cells, which has a poor prognosis and high mortality. Consequently, effective therapeutic options are required to improve the treatment outcome. In the present study, we demonstrated the effects of genistein on sensitizing CCA and enhancing natural killer (NK-92) cells to induce cytotoxicity of CCA cells. Genistein treatment could promote extrinsic apoptotic pathway to induce CCA cell death. Mechanistically, genistein treatment decreased the expression of anti-apoptotic protein, cFLIP, and significantly upregulated the expression of death receptors (Fas receptor; FasR, and TRAIL receptor; TRAIL-R), which might contribute to the increased susceptibility of CCA to NK-92 cells.Cholangiocarcinoma (CCA) is a lethal bile duct cancer, which has poor treatment outcomes due to its high resistance to chemotherapy and cancer recurrence. Activation of aberrant anti-apoptotic signaling pathway has been reported to be a mechanism of chemoresistance and immune escape of CCA. Therefore, reversal of anti-apoptotic signaling pathway represents a feasible approach to potentiate effective treatments, especially for CCA with high chemoresistance. In this study, we demonstrated the effects of genistein on reactivation of apoptosis cascade and increase the susceptibility of CCA cells to natural killer (NK-92) cells. Genistein at 50 and 100 µM significantly activated extrinsic apoptotic pathway in CCA cells (KKU055, KKU100, and KKU213A), which was evident by reduction of procaspase-8 and -3 expression. Pretreatment of CCA cells with genistein at 50 µM, but not NK-92 cells, significantly increased NK-92 cell killing ability over the untreated control, suggesting the ability of genistein to sensitize CCA cells. Interestingly, genistein treatment could greatly lower the expression of cFLIP, an anti-apoptotic protein involved in the immune escape pathway, in addition to upregulation of death receptors, Fas- and TRAIL-receptors, in CCA cells, which might be the underlying molecular mechanism of genistein to sensitize CCA to be susceptible to NK-92 cells. Taken together, this finding revealed the benefit of genistein as a sensitizer to enhance the efficiency of NK cell immunotherapy for CCA.

Baicalin Induced Apoptosis of Human Cholangiocarcinoma Cell through Activating AMPK/mTORC1/p70S6K Signaling Pathway.

Baicalin (naturally bioactive flavone compound isolated from Scutellaria baicalensis) has been demonstrated to exert strong anticancer activity against various tumor cells. However, the possibility of using baicalin for the treatment of cholangiocarcinoma and its effectiveness remain unstudied. The effect of baicalin on QBC939 cholangiocarcinoma cell culture was studied by assessing cell viability (CCK-8 test) and expression of the key proteins (Western blotting). Baicalin induced apoptosis of QBC939 cells in culture in a dose- and time-dependent manner. The proapoptotic effect was attributed to inhibition of the mTORC1-p70S6K signaling pathway resulting from baicalin-induced AMPK activation. These findings provide a new approach for cholangiocarcinoma treatment and serve as a basis for developing baicalin-based combination cancer therapy strategies.

The Casein Kinase 2 Inhibitor CX-4945 Promotes Cholangiocarcinoma Cell Death Through PLK1.

Casein Kinase 2 (CK2) is a prosurvival protein kinase involved in cell growth/proliferation through the regulation of the cell cycle and apoptosis. CK2 is over-expressed in various cancers, which correlates with a poor prognosis. This study examined the anti-cancer effects of silmitasertib (CX-4945), a CK2 inhibitor, on cholangiocarcinoma (CCA) cells. The effects of CX-4945 on cell viability, cell cycle arrest, and apoptosis in the human cholangiocarcinoma cell lines TFK-1 and SSP-25 were evaluated. Alterations in posttranslational modifications and the levels of cell cycle regulators including p21, Polo-like kinase 1 (PLK1), andp53 were assessed by western blotting. Apoptotic responses were examined using Propidium iodine/Annexin V staining. TFK-1 and SSP-25 cells exposed to CX-4945 showed morphologic changes and a more than 50% decrease in cell viability (p<0.05). Cell cycle arrest at the G2 phase was detected following an increase in phosphorylated PLK1 and p21. Furthermore, phospho-PLK1 induced the degradation of p53, which led to the dissociation of Bax from Bcl-xL. The cleavage of Caspase3 and PARP were also induced by CX-4945 treatment. CX-4945 induces cell cycle arrest and cell death in cholangiocarcinoma cells via the regulation of PLK1 and p53. This may provide a novel therapeutic strategy for advanced cholangiocarcinoma.

Novel Nitric Oxide Donor Dinitroazetidine-Coumarin Hybrids as Potent Anti-Intrahepatic Cholangiocarcinoma Agents.

Intrahepatic cholangiocarcinoma (iCC) is a serious liver cancer threatening human health. However, there are a few chemotherapeutic drugs for the treatment of iCC in the clinic. It is extremely urgent to develop new drugs for iCC. In this study, twenty dinitroazetidine and coumarin hybrids were synthesized and evaluated anti-iCC bioactivity as a new type of nitric oxide (NO) donors. Among them, compounds 2–5 and 21 showed a higher antiproliferative activity against RBE cell lines (human intrahepatic cholangiocarcinoma cell lines) and low cytotoxicity in nontumor cells (HOSEpiC and T29). The preliminary study of pharmacology mechanism indicated that compounds 2–5 and 21 could release effective concentration of NO in RBE cell lines, which leaded to inhibit the proliferation of RBE cell lines. The research results revealed that compound 3 inhibited the proliferation of RBE cell lines by inducing apoptosis and arresting cell cycle at G2/M phase. Additionally, compound 3 had acceptable metabolic stability. Therefore, compound 3 was merited to further explore for developing a desirable NO donor lead with anti-iCC activity.

Zic family member 5 promotes survival in human pancreatic cancer and cholangiocarcinoma cells

Pancreatic ductal adenocarcinoma (PDAC) and cholangiocarcinoma (CCA) are malignant tumors with poor prognosis because of the limited effectiveness of traditional chemotherapy and few effective molecular therapeutic agents. Here, we determined the essential roles of Zic family member 5 (ZIC5) in the survival of PDAC and CCA cells. The results showed that ZIC5 is strongly expressed in PDAC and CCA tissues, while ZIC5 expression is barely observed in most normal human adult tissues. Furthermore, ZIC5 expression is related to poor prognosis of patients with PDAC. ZIC5 knockdown via small interfering RNA decreased the phosphorylation of signal transducer and activator of transcription 3 (STAT3), a protein that is associated with PDAC and CCA aggressiveness. However, ZIC5 knockdown induced cell death regardless of STAT3 activation, which is promoted by interleukin (IL) −6, a factor associated with inflammation. Furthermore, knockdown of ZIC5 in PDAC and CCA cells additively or synergistically induced apoptosis with the anti-cancer drug gemcitabine. Thus, ZIC5 constitutes a potential therapeutic target for the treatment of PDAC and CCA.

Deciphering genomic characteristics of ERBB family members potentially involved in the recovery of anti-cancer immunity for intrahepatic cholangiocarcinoma patients.

e16003 Background: Previous studies showed that the efficacy of immune checkpoint inhibitor (ICI) is enhanced in hepatobiliary cancers harboring activating gene alterations of ERBB family members. The purpose of this study was to explore genomic characteristics of ERBB family members as well as the relationship of them with the activity of anti-cancer immunity in Chinese intrahepatic cholangiocarcinoma (ICC) patients. Methods: Fifty-four ICC patients which carried at least one gene mutation of ERBB family members were pre-selected from 508 ICC patients included in the China Pan-cancer project of OrigiMed 2020 (https://www.cbioportal.org/study/summary?id=pan_origimed_2020). Targeted sequencing was implemented with cancer-related 450-gene panel. Tumor mutation burden (TMB) and microsatellite status were evaluated. To explore the molecular mechanisms, two human cholangiocarcinoma cell lines, RBE and HuCCT1, were transfected with a lentivirus to obtain the cells with stable S310F mutation and overexpression of ERBB2. Results: The variation frequencies of EGFR, ERBB2, ERBB3 and ERBB4 were 2.8% (14/508), 3.5% (18/508), 3.3% (17/508) and 2.4% (12/508) in Chinese ICC patients, respectively. All the patients harboring ERBB family gene mutations were classified as TMB-H (cutoff = 10 Muts/Mb), when co-mutated with ACVR2A or TGFBR2. Moreover, ACVR2A and TGFBR2 were identified as driver genes in the cohort with oncodriveCLUST. TMB and the proportion of MSI-H patients were significantly elevated when ERBB family members co-mutated with ACVR2A (mutation vs wild: TMB, 38.2 ± 19.1 vs 7.0 ± 8.6 Muts/Mb, p&lt;0.0001; MSI-H, 77.8% vs 4.9%, p&lt;0.0001), or with TGFBR2 (TMB, 33.1 ± 8.7 vs 9.0 ± 14.4 Muts/Mb, p&lt;0.0001; MSI-H, 83.3% vs 9.1%, p&lt;0.001). Besides, ACVR2A and TGFBR2 were enriched in TGF-β pathway. Flow cytometry analysis revealed that ERBB2 S310F mutation and amplification were increased by approximate 3-fold and 3.5-fold in PD-L1 expression of cell line RBE ( p &lt; 0.0001, respectively); by approximate 2-fold and 3-fold upregulation of PD-L1 expression in cell line HUCCT1 ( p &lt; 0.0001, respectively). Western-blot results suggested that ERBB2 mutation/overexpression mediated PD-L1 upregulation through the RAS/ERK/c-JUN pathway. T cell cytotoxicity-mediated killing assay demonstrated that ERBB2 S310F mutation and overexpression induced the recovery of anti-cancer immunity by about 30% ( p &lt; 0.05) both in RBE and HuCCT1 cells. Conclusions: The present study revealed two distinct genomic characteristics potentially involved in the recovery of anti-cancer immunity of intrahepatic cholangiocarcinoma patients: on one hand, co-mutation of ERBB family genes with ACVR2A or TGFBR2 was enriched in TGF-β pathway, contributing to MSI-H/TMB-H; on the other hand, ERBB2 might impact expression of PD-L1 by regulating the RAS/ERK/c-Jun pathways.

PH and Redox-Dual Sensitive Chitosan Nanoparticles Having Methyl Ester and Disulfide Linkages for Drug Targeting against Cholangiocarcinoma Cells.

The aim of this study is to prepare pH- and redox-sensitive nanoparticles for doxorubicin (DOX) delivery against DOX-resistant HuCC-T1 human cholangiocarcinoma (CCA) cells. For this purpose, L-histidine methyl ester (HIS) was attached to chitosan oligosaccharide (COS) via dithiodipropionic acid (abbreviated as ChitoHISss). DOX-incorporated nanoparticles of ChitoHISss conjugates were fabricated by a dialysis procedure. DOX-resistant HuCC-T1 cells were prepared by repetitive exposure of HuCC-T1 cells to DOX. ChitoHISss nanoparticles showed spherical morphology with a small diameter of less than 200 nm. The acid pH and glutathione (GSH) addition induced changes in the size distribution pattern of ChitoHISss nanoparticles from a narrow/monomodal distribution pattern to a wide/multimodal pattern and increased the fluorescence intensity of the nanoparticle solution. These results indicate that a physicochemical transition of nanoparticles can occur in an acidic pH or redox state. The more acidic the pH or the higher the GSH concentration the higher the drug release rate was, indicating that an acidic environment or higher redox states accelerated drug release from ChitoHISss nanoparticles. Whereas free DOX showed decreased anticancer activity at DOX-resistant HuCC-T1 cells, DOX-incorporated ChitoHISss nanoparticles showed dose-dependent anticancer activity. Intracellular delivery of DOX-incorporated ChitoHISss nanoparticles was relatively increased at an acidic pH and in the presence of GSH, indicating that DOX-incorporated ChitoHISss nanoparticles have superior acidic pH- and redox-sensitive behavior. In an in vivo tumor xenograft model, DOX-incorporated ChitoHISss nanoparticles were specifically delivered to tumor tissues and then efficiently inhibited tumor growth. We suggest that ChitoHISss nanoparticles are a promising candidate for treatment of CCA.