Saikosaponin D reverses epinephrine- and norepinephrine-induced gemcitabine resistance in intrahepatic cholangiocarcinoma by downregulating ADRB2/glycolysis signaling.

Intrahepatic cholangiocarcinoma (iCCA) is a highly fatal malignancy with rapidly increasing incidence and mortality worldwide. Currently, gemcitabine-based systemic chemotherapy is the main clinical therapeutic regimen; however, its efficacy is poor, and its mechanism has not been elucidated. In this study, we use a Seahorse Extracellular Flux analyser to measure glycolysis capacity (extracellular acidification rate, ECAR) and oxygen consumption rate (OCR). The glucose uptake or lactic acid content is detected, and the effects of saikosaponin D, an active compound derived from Bupleuri Radix (a traditional Chinese medicine for soothing the liver and relieving depression), on gemcitabine cytotoxicity in norepinephrine-stimulated iCCA cells are analysed. We find that adrenergic signaling plays a fundamental role in chronic stress-induced therapeutic resistance in iCCA. Norepinephrine (NE) and epinephrine (E) enhance the proliferation of iCCA cells and interfere with the response to gemcitabine through activation of the ß2-adrenergic receptor (ADRB2). Furthermore, we find that NE upregulates the expressions of several drug efflux-related genes (such as ABCG2 and MDR1) and promotes glycolysis in iCCA cells. In addition, saikosaponin D reverses the poor response of iCCA cells to gemcitabine by downregulating ADRB2 level. Furthermore, saikosaponin D inhibits drug efflux and glycolysis in iCCA cells by regulating the expressions of MDR1, ABCG2, HK2, and GLUT1. Collectively, saikosaponin D enhances the antitumor effect of gemcitabine by controlling glucose metabolism and drug efflux by inhibiting the ADRB2 signaling. Therefore, the combination of saikosaponin D and gemcitabine may be a potential therapeutic strategy for the treatment of iCCA.

Quercetin Inhibits Intrahepatic Cholangiocarcinoma by Inducing Ferroptosis and Inhibiting Invasion via the NF-[Formula: see text]B Pathway.

Intrahepatic cholangiocarcinoma (ICC) is a rare, highly fatal hepatobiliary malignancy, with very limited treatment options and, consequently, a poor prognosis. Recently, emerging evidence has suggested the potential of quercetin (QE) for use in cancer therapy. The purpose of this study is to investigate whether QE could inhibit ICC. The effects of QE on the proliferation, apoptosis, and invasion of ICC were analyzed in vitro. The inhibitory effect of QE on ICC was also verified in vivo. The RNA sequence was applied to explore the mechanism of QE. Functional verification was also performed after RNA sequencing using activators and inhibitors of nuclear factor-kappa-B (NF-[Formula: see text]B) and ferroptosis. The results showed that QE could inhibit the proliferation and survival of ICC cells, induce the arrest of ICC cells in the G1 phase, promote the apoptosis of ICC cells, and inhibit the invasion of ICC cells. Furthermore, QE could promote ferroptosis in ICC cells by inhibiting the NF-[Formula: see text]B pathway. In conclusion, QE is a new ferroptosis inducer and NF-[Formula: see text]B inhibitor that can not only induce ferroptosis, but also inhibit the invasion of ICC cells, providing a prospective strategy for the treatment of ICC.

Human Metastatic Cholangiocarcinoma Patient-Derived Xenografts and Tumoroids for Preclinical Drug Evaluation.

PURPOSE: Cholangiocarcinoma (CCA) is usually diagnosed at advanced stages, with limited therapeutic options. Preclinical models focused on unresectable metastatic CCA are necessary to develop rational treatments. Pathogenic mutations in IDH1/2, ARID1A/B, BAP1, and BRCA1/2 have been identified in 30%-50% of patients with CCA. Several types of tumor cells harboring these mutations exhibit homologous recombination deficiency (HRD) phenotype with enhanced sensitivity to PARP inhibitors (PARPi). However, PARPi treatment has not yet been tested for effectiveness in patient-derived models of advanced CCA. EXPERIMENTAL DESIGN: We have established a collection of patient-derived xenografts from patients with unresectable metastatic CCA (CCA_PDX). The CCA_PDXs were characterized at both histopathologic and genomic levels. We optimized a protocol to generate CCA tumoroids from CCA_PDXs. We tested the effects of PARPis in both CCA tumoroids and CCA_PDXs. Finally, we used the RAD51 assay to evaluate the HRD status of CCA tissues. RESULTS: This collection of CCA_PDXs recapitulates the histopathologic and molecular features of their original tumors. PARPi treatments inhibited the growth of CCA tumoroids and CCA_PDXs with pathogenic mutations of BRCA2, but not those with mutations of IDH1, ARID1A, or BAP1. In line with these findings, only CCA_PDX and CCA patient biopsy samples with mutations of BRCA2 showed RAD51 scores compatible with HRD. CONCLUSIONS: Our results suggest that patients with advanced CCA with pathogenic mutations of BRCA2, but not those with mutations of IDH1, ARID1A, or BAP1, are likely to benefit from PARPi therapy. This collection of CCA_PDXs provides new opportunities for evaluating drug response and prioritizing clinical trials.

YY1-induced DLEU1/miR-149-5p Promotes Malignant Biological Behavior of Cholangiocarcinoma through Upregulating YAP1/TEAD2/SOX2.

Cholangiocarcinoma is an extremely malignant cancer with poor prognosis. Finding efficient diagnosis and treatment is the indispensable way to improve the prognosis of CCA patients. Therefore, exploring molecular abnormalities in CCA development is urgently needed. DLEU1 is a potential tumor-related lncRNA and abnormally expressed in multiple cancers. In this study, TCGA data analysis showed upregulation of DLEU1 expression in CCA. Furthermore, we confirmed that DLEU1 expression was increased in CCA tissues and cells compared with corresponding controls. Upregulated DLEU1 was related to poor clinicopathological characteristics. Functionally, silencing DLEU1 inhibited CCA proliferation, invasion, stemness maintenance and chemo-resistance, whereas amplifying DLEU1 promoted malignant biological behavior of CCA cells. Mechanistically, DLEU1 expression was transcriptionally facilitated by transcription factor YY1. Moreover, DLEU1 promoted oncogene YAP1 expression by functioning as a sponge to competitively bind to miR-149-5p. YAP1 promoted CCA proliferation, invasion and stemness maintenance, whereas miR-149-5p inhibited malignant biological behavior of CCA. Rescue experiments confirmed that the cancer-promoting effect of DLEU1 was saved by interfering miR-149-5p or YAP1. Furthermore, YAP1 promoted tumor stemness maintenance partly by acting as a transcriptional coactivator to promote TEAD2-induced SOX2 expression. These findings indicated that YY1-induced DLEU1 played a crucial role in CCA progression via miR-149-5p/YAP1/TEAD2/SOX2 axis.

Successful Treatment of Advanced Intrahepatic Cholangiocarcinoma With a High Tumor Mutational Burden and PD-L1 Expression by PD-1 Blockade Combined With Tyrosine Kinase Inhibitors: A Case Report.

Advanced intrahepatic cholangiocarcinoma (iCCA) is not suitable for surgical treatment. Guided by the concept of precision medicine, preoperative systematic treatment may reshape the clinical outcomes of advanced intrahepatic cholangiocarcinoma patients. We describe the case of a 38-year-old female who has been diagnosed with stage IV intrahepatic cholangiocarcinoma with a high tumor mutational burden and positively programmed death-ligand 1 (PD-L1) expression. The patient was treated with programmed cell death 1 (PD-1) inhibitors combined with tyrosine kinase inhibitors (TKIs). After 7 cycles of combination therapy, she underwent radical resection and no tumor cells were found in the postoperative histopathological examination. In addition, the patient's survival time had reached 25 months, as of August 2021. To date, this is the first case of successful radical resection after combined immunotherapy with TKIs for advanced PD-L1-positive intrahepatic cholangiocarcinoma with a high tumor mutational burden (TMB). The case provides a new approach to the treatment of advanced intrahepatic cholangiocarcinoma.

Circ-LAMP1 contributes to the growth and metastasis of cholangiocarcinoma via miR-556-5p and miR-567 mediated YY1 activation.

Dysregulation of circular RNAs (circRNAs) executes important regulatory roles in carcinogenesis. Nonetheless, few studies focused on the mechanisms of circRNAs in cholangiocarcinoma (CCA). qRT-PCR was applied to verify the dysregulated circRNAs in CCA. Fisher's exact test, Kaplan-Meier analysis and Cox regression model were utilized to investigate the clinical implications of circ-LAMP1 in the patients with CCA. The viability, apoptosis, migration and invasion of CCA cells were detected after silencing/overexpression of circ-LAMP1. Xenograft and lung metastasis assays were performed to verify the in vitro results. The regulatory networks of circ-LAMP1 were unveiled by bioinformatic analysis, RNA immunoprecipitation (RIP), RNA pulldown and luciferase reporter assays. Up-regulation of circ-LAMP1 was found in CCA tissue samples and cell lines. Enhanced level of circ-LAMP1 was linked to clinical severity, high post-operative recurrence and poor prognosis for the patients with CCA. Gain/loss-of-function assays confirmed the oncogenic role of circ-LAMP1 in mediating cell growth, apoptosis, migration and invasion. Nevertheless, the level of circ-LAMP1 had no effect on normal biliary epithelium proliferation and apoptosis. Animal study further verified the in vitro data. Mechanistically, circ-LAMP1 directly sponged miR-556-5p and miR-567, thereby releasing their suppression on YY1 at post-transcriptional level. Rescue assay indicated that the oncogenic role of circ-LAMP1 is partially dependent on its modulation of YY1 in CCA. In summary, this study suggested that circ-LAMP1 might be used as a promising biomarker/therapeutic target for CCA.

A New Option for the Treatment of Intrahepatic Cholangiocarcinoma: Percutaneous Hepatic Perfusion with CHEMOSAT Delivery System.

Liver metastases are a major management problem; since they occur in tumors of different origin, they are often multiple, difficult to visualize and can lie dormant for many years. Patients with liver metastases usually die of their disease, mostly due to liver failure, since systemic treatments are unable to eradicate micro-metastasis, and interventional loco-regional procedures cannot treat all existing ones. Cholangiocarcinoma (CCA) is the second most common primary liver tumor, showing a poor overall prognosis. When resection is not possible, treatment options include tumor-focused or local ablative therapy, organ-focused or regional therapy and systemic therapy. We reviewed available loco-regional therapeutic options, with particular focus on the CHEMOSAT® Melphalan/Hepatic Delivery System (CS-HDS), which is uniquely positioned to perform a percutaneous hepatic perfusion (PHP), in order to treat the entire liver as a standalone or as complementary therapy. This system isolates the liver circulation, delivers a high concentration of chemotherapy (melphalan), filters most chemotherapy out of the blood and is a repeatable procedure. Most CS-HDS benefits are demonstrated in liver-predominant diseases, like liver metastasis from uveal melanoma (UM), hepatocarcinoma (HCC) and CCA. More than 650 procedures have been performed in Europe to date, mostly to treat liver metastases from UM. In CCA, experience is still limited, but retrospective analyses have been reported, while phase II and III studies are closed, waiting for results or ongoing.

A Circular RNA, Cholangiocarcinoma-Associated Circular RNA 1, Contributes to Cholangiocarcinoma Progression, Induces Angiogenesis, and Disrupts Vascular Endothelial Barriers.

BACKGROUND AND AIMS: Circular RNAs (circRNAs) and extracellular vesicles (EVs) are involved in various malignancies. We aimed to clarify the functions and mechanisms of dysregulated circRNAs in the cells and EVs of cholangiocarcinoma (CCA). APPROACH AND RESULTS: CircRNA microarray was used to identify circRNA expression profiles in CCA tissues and bile-derived EVs (BEVs). CCA-associated circRNA 1 (circ-CCAC1) expression was measured by quantitative real-time PCR. The clinical importance of circ-CCAC1 was analyzed by receiver operating characteristic curves, Fisher's exact test, Kaplan-Meier plots, and Cox regression model. The functions of circ-CCAC1 and exosomal circ-CCAC1 were explored in CCA cells and human umbilical vein endothelial cells (HUVECs), respectively. Different animal models were used to verify the in vitro results. RNA sequencing, bioinformatics, RNA immunoprecipitation, RNA pulldown, chromatin immunoprecipitation followed by sequencing, and luciferase reporter assays were used to determine the regulatory networks of circ-CCAC1 in CCA cells and HUVECs. Circ-CCAC1 levels were increased in cancerous bile-resident EVs and tissues. The diagnostic and prognostic values of circ-CCAC1 were identified in patients with CCA. For CCA cells, circ-CCAC1 increased cell progression by sponging miR-514a-5p to up-regulate Yin Yang 1 (YY1). Meanwhile, YY1 directly bound to the promoter of calcium modulating ligand to activate its transcription. Moreover, circ-CCAC1 from CCA-derived EVs was transferred to endothelial monolayer cells, disrupting endothelial barrier integrity and inducing angiogenesis. Mechanistically, circ-CCAC1 increased cell leakiness by sequestering enhancer of zeste homolog 2 in the cytoplasm, thus elevating SH3 domain-containing GRB2-like protein 2 expression to reduce the levels of intercellular junction proteins. In vivo studies further showed that increased circ-CCAC1 levels in circulating EVs and cells accelerated both CCA tumorigenesis and metastasis. CONCLUSIONS: Circ-CCAC1 plays a vital role in CCA tumorigenesis and metastasis and may be an important biomarker/therapeutic target for CCA.

Yin Yang 1-induced LINC00667 up-regulates pyruvate dehydrogenase kinase 1 to promote proliferation, migration and invasion of cholangiocarcinoma cells by sponging miR-200c-3p.

Cholangiocarcinoma (CCA) is one of the most aggressive and lethal malignancies. Long noncoding RNAs (lncRNAs) are being found to play crucial roles in CCA progression. This work aims to investigate the roles of long intergenic non-protein coding RNA 667 (LINC00667) in progression of CCA. RT-qPCR and western blot were applied to detect gene expression. Clinical correlation and survival were analyzed by statistical methods. Overexpression and RNA interference approaches were used to investigate the effects of LINC00667 on CCA cells. Tumor xenograft assay was performed to detect the function of LINC00667 in vivo. Transcriptional regulation and competing endogenous RNA (ceRNA) mechanism were predicted via bioinformatics analysis. ChIP, luciferase reporter, and Ago2 RIP assays further confirmed the predicted results. Our data indicated that LINC00667 was highly expressed in CCA tissues and cells, and transcription factor Yin Yang 1 (YY1) induced LINC00667 expression in CCA cells. Up-regulated LINC00667 was significantly associated with lymph node metastasis, advanced TNM stage, and poor prognosis. Knockdown of LINC00667 suppressed the proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of CCA cells, while overexpression of LINC00667 acquired opposite effects. Moreover, knockdown of LINC00667 inhibited tumor growth in vivo. In addition, LINC00667 was demonstrated to function as a ceRNA for miR-200c-3p, and then LINC00667 up-regulated pyruvate dehydrogenase kinase 1 (PDK1) to promote CCA development by inhibiting miR-200c-3p. These findings identified a pivotal role of LINC00667 in tumorigenesis and development of CCA. Targeting the YY1/LINC00667/miR-200c-3p/PDK1 axis may provide a new therapeutic strategy for CCA treatment.

Targeted Modulation of FAK/PI3K/PDK1/AKT and FAK/p53 Pathways by Cucurbitacin B for the Antiproliferation Effect Against Human Cholangiocarcinoma Cells.

Inadequate responses to traditional chemotherapeutic agents in cholangiocarcinoma (CCA) emphasize a requirement for new effective compounds for the treatment of this malignancy. This study aimed to investigate the antiproliferative property of cucurbitacin B on KKU-100 CCA cells. The determination of underlying molecular mechanisms was also carried out. The results revealed that cucurbitacin B suppressed growth and replicative ability to form colonies of CCA cells, suggesting the antiproliferative effect of this compound against the cells. Flow cytometry analysis demonstrated that the interfering effect of cucurbitacin B on the CCA cell cycle at the G2/M phase was accountable for its antiproliferation property. Accompanied with cell cycle disruption, cucurbitacin B altered the expression of proteins involved in the G2/M phase transition including downregulation of cyclin A, cyclin D1, and cdc25A, and upregulation of p21. Additional molecular studies demonstrated that cucurbitacin B suppressed the activation of focal adhesion kinase (FAK) which consequently resulted in inhibition of its kinase-dependent and kinase-independent downstream targets contributing to the regulation of cell proliferation including PI3K/PDK1/AKT and p53 proteins. In this study, the transient knockdown of FAK using siRNA was employed to ascertain the role of FAK in CCA cell proliferation. Finally, the effect of cucurbitacin B on upstream receptor tyrosine kinases regulating FAK activation was elucidated. The results showed that the inhibitory effect of cucurbitacin B on FAK activation in CCA cells is mediated via interference of EGFR and HER2 expression. Collectively, cucurbitacin B might be a promising drug for CCA treatment by targeting FAK protein.

Prolonged Response to Anti-PD-1 Antibody Therapy in Chemotherapy-Refractory Cholangiocarcinoma With High Tumor Mutational Burden.

Management of advanced intrahepatic cholangiocarcinoma (iCCA) is challenging and overall survival is poor. Progress in the development of new therapeutic options for metastatic cholangiocarcinoma (CCA) has been slow; hence, to date, there are no approved second-line agents in this setting. Although the development of immune checkpoint inhibitors has significantly improved overall survival in a variety of malignancies, there has not been a clinically important impact in CCA. This report presents a 66-year-old patient with chemotherapy-refractory iCCA who experienced a prolonged response to immunotherapy. Tumor genome profiling revealed a high tumor mutation burden of 17 mutations per megabase in the absence of microsatellite instability. He was started on immunotherapy with nivolumab and has experienced an ongoing response for 16 months without clinical symptoms and only minimal radiologic disease.

Causes of hOCT1-Dependent Cholangiocarcinoma Resistance to Sorafenib and Sensitization by Tumor-Selective Gene Therapy.

Although the multi-tyrosine kinase inhibitor sorafenib is useful in the treatment of several cancers, cholangiocarcinoma (CCA) is refractory to this drug. Among other mechanisms of chemoresistance, impaired uptake through human organic cation transporter type 1 (hOCT1) (gene SLC22A1) has been suggested. Here we have investigated the events accounting for this phenotypic characteristic and have evaluated the interest of selective gene therapy strategies to overcome this limitation. Gene expression and DNA methylation of SLC22A1 were analyzed using intrahepatic (iCCA) and extrahepatic (eCCA) biopsies (Copenhagen and Salamanca cohorts; n = 132) and The Cancer Genome Atlas (TCGA)-CHOL (n = 36). Decreased hOCT1 mRNA correlated with hypermethylation status of the SLC22A1 promoter. Treatment of CCA cells with decitabine (demethylating agent) or butyrate (histone deacetylase inhibitor) restored hOCT1 expression and increased sorafenib uptake. MicroRNAs able to induce hOCT1 mRNA decay were analyzed in paired samples of TCGA-CHOL (n = 9) and Copenhagen (n = 57) cohorts. Consistent up-regulation in tumor tissue was found for miR-141 and miR-330. High proportion of aberrant hOCT1 mRNA splicing in CCA was also seen. Lentiviral-mediated transduction of eCCA (EGI-1 and TFK-1) and iCCA (HuCCT1) cells with hOCT1 enhanced sorafenib uptake and cytotoxic effects. In chemically induced CCA in rats, reduced rOct1 expression was accompanied by impaired sorafenib uptake. In xenograft models of eCCA cells implanted in mouse liver, poor response to sorafenib was observed. However, tumor growth was markedly reduced by cotreatment with sorafenib and adenoviral vectors encoding hOCT1 under the control of the BIRC5 promoter, a gene highly up-regulated in CCA. Conclusion: The reason for impaired hOCT1-mediated sorafenib uptake by CCA is multifactorial. Gene therapy capable of selectively inducing hOCT1 in tumor cells can be considered a potentially useful chemosensitization strategy to improve the response of CCA to sorafenib.

Actinidia arguta (Hardy Kiwi) Root Extract Exerts Anti-cancer Effects via Mcl-1-Mediated Apoptosis in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a highly aggressive and chemoresistant liver malignancy. Thus, identification of strategies to overcome insensitivity to apoptosis and growth inhibition is a growing focus of research in this malignancy. This study evaluated the potential anti-cancer effects of an ethanol extract from the Actinidia arguta (Hardy Kiwi) root (RAE) on CCA. Our data demonstrated that RAE decreased cell viability and induced apoptosis by activation of Caspase 3, Caspase 8, and Poly (ADP-ribose) polymerase (PARP) in two CCA cell lines. RAE induced a decrease in Mcl-1 in cultured CCA cells and in xenograft CCA tumors. Administration of RAE every other day led to significant growth inhibition in tumor burden xenograft CCA mice. Western blotting analysis of paired human CCA and normal adjacent tissues from the same patient revealed that CCA tissues exhibited significantly higher Mcl-1 expression than normal tissues. Taken together, our findings demonstrated the anti-cancer effects of RAE on CCA both in vitro and in vivo. These data suggest that RAE may be a promising anti-CCA agent and could be beneficial in the treatment of CCA through the targeting of Mcl-1.

Anti-cancer activity of asiatic acid against human cholangiocarcinoma cells through inhibition of proliferation and induction of apoptosis.

Plant-derived anti-cancer agents have been of considerable interest due to their promising effectiveness with low side effects. Asiatic acid, the main constituent of the medicinal plant Centella asiatica (L.) Urban, has a wide range of biological properties such as antioxidant, anti-inflammatory and anti-cancer activities. Cholangiocarcinoma (CCA), which is a malignant tumor of bile duct epithelium, is one of the leading cancers in Southeast Asia, notably the northeast of Thailand where the liver fluke, Opisthorchis viverrini predominates. Many in vitro and in vivo studies have provided evidence supporting that oxidative stress induced by chronic inflammation is involved in CCA genesis with aggressive clinical outcomes. This study was performed to evaluate the cytotoxic effects of asiatic acid on two human CCA cell lines (KKU-156 and KKU-213). Cell viability was determined by a sulforhodamine B (SRB) assay. Morphological changes of the cells were observed by microscopy. Cell apoptosis was detected by flow cytometry using annexin V and propidium iodide (PI) staining. Messenger RNA (mRNA) expression levels of BAX, BCL2 and Survivin/BIRC5 were analyzed by real-time polymerase chain reaction (PCR). It was found that asiatic acid efficiently suppressed CCA cellular viability via induction of apoptosis. In addition, the occurrence of asiatic acid-induced apoptosis was confirmed by microscopic observation of apoptotic vesicles, down-regulation of anti-apoptotic genes (BCL2 and Survivin/BIRC5) and increased early and late apoptotic cells. Our results showed the chemotherapeutic activities of asiatic acid, suggesting the anti-cancer properties of this compound should be clinically assessed and its supplementation may lead to an improvement of survival of CCA patients.

Dihydroartemisinin induces apoptosis and autophagy-dependent cell death in cholangiocarcinoma through a DAPK1-BECLIN1 pathway.

Cholangiocarcinoma (CCA) is a very aggressive cancer arising from the malignant transformation of cholangiocytes. Intrahepatic CCA is associated with reactive inflammation and intense fibrosis of the hepatobiliary tract. Dihydroartemisinin (DHA), the active compound found in Artemisia annua, has been shown to possess anti-tumor activity in a variety of human cancers, including hepatoma. Here, we tested the ability of DHA to specifically kill CCA cells and have investigated the underlying mechanisms. DHA induced both apoptosis and autophagy-dependent caspase-independent cell death in many CCA cell lines, while being slightly toxic to immortalized cholangiocytes. DHA induced the expression of many apoptosis- and autophagy-related genes in CCA cells. In particular, it greatly induced the expression of DAPK1, and reduced the interaction of BECLIN1 with BCL-2 while promoting its interaction with PI3KC3. Genetic silencing of DAPK1 prevented DHA-induced autophagy. Pharmacologic and genetic inhibition of BECLIN1 function prevented autophagy and cell death induced by DHA in CCA cells. These data unravel a novel pathway of DHA cancer toxicity and open the possibility to introduce DHA in the therapeutic regimen for the treatment of CCA.

Cratoxylum formosum Extracts Inhibit Growth and Metastasis of Cholangiocarcinoma Cells by Modulating the NF-κB and STAT3 Pathways.

Cratoxylum formosum Dyer has been used in Southeast Asian countries both for food and folk medicine. In this study, the leaf extracts of C. formosum were evaluated for anticancer effects on human cholangiocarcinoma (CCA) KKU-M156 cells. The results showed that the plant extracts possessed potent cytotoxicity against CCA cells. The cytotoxic activity was associated with an induction of cell apoptosis. Moreover, the colony forming ability of CCA cells was also inhibited by C. formosum extracts. Consistent with growth inhibitory effects, the plant extracts induced cell cycle arrest at the G2/M phase and downregulated cyclin A and Cdc25A protein expression. The extracts potently suppressed the migration and invasion properties of CCA cells. The effects were associated with the suppression of NF-κB and STAT3 nuclear translocation and transcriptional activity, and downregulation of genes involving in cancer progression and metastasis. Furthermore, the possible bioactive compounds in the extracts were analyzed by HPLC. Taken together, the potent anticancer activity of C. formosum against CCA indicates the plant promising use for CCA prevention and therapy.

Gene expression-based chemical genomics identifies heat-shock protein 90 inhibitors as potential therapeutic drugs in cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma (CCA) is an aggressive tumor with a poor prognosis. There is no standard therapy for CCA, and novel drugs for treating refractory CCA need to be identified. METHODS: The authors hypothesized that, if a drug could reverse the gene expression signature of CCA, then it may inhibit the carcinogenesis of CCA and, hence, would be a potential therapeutic agent. Thus, the gene expression signatures from patients with CCA were queried using the bioinformatic method Connectivity Map, resulting in the enrichment of heat-shock protein 90 (HSP90) inhibitors with therapeutic potentials. RESULTS: Two HSP90 inhibitors, 17-AAG (tanespimycin) and the synthetic diarylisoxazole amide resorcinol NVP-AUY922, demonstrated potent antiproliferative activity in in vitro studies. In a thioacetamide-induced animal model, NVP-AUY922 also had antitumor activity and resulted in objective tumor regression. In addition, NVP-AUY922 reduced the expression of client oncoproteins involved in CCA oncogenesis and inhibited downstream proteins of both the phosphatidylinositol 3-kinase catalytic subunit α/v-akt murine thymoma viral oncogene homolog 1 protein kinase (PIK3/AKT) pathway and the v-Ki-ras2 Kirsten rat sarcoma viral oncogene/mitogen-activated protein kinase (KRAS/MAPK) pathway. CONCLUSIONS: Preclinical data from the current study suggest that NVP-AUY922 may be an effective treatment option for patients with CCA.

Cyclooxygenase-2-derived prostaglandin E2 activates beta-catenin in human cholangiocarcinoma cells: evidence for inhibition of these signaling pathways by omega 3 polyunsaturated fatty acids.

Cholangiocarcinoma is a highly malignant neoplasm of the biliary tree. It has a high rate of mortality, and currently, there is no effective chemoprevention and treatment. This study was designed to investigate the potential effect of omega 3 polyunsaturated fatty acids (omega 3-PUFA) on human cholangiocarcinoma cell growth and to determine their mechanisms of actions. Treatment of three human cholangiocarcinoma cells (CCLP1, HuCCT1, SG231) with two omega 3-PUFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), for 12 to 72 h resulted in a dose- and time-dependent inhibition of cell growth; in contrast, arachidonic acid, a omega 6-PUFA, had no significant effect. The omega 3-PUFA effect is due to the induction of apoptosis, given that DHA induced the cleaved form of PARP, caspase-3, and caspase-9. DHA and EPA treatment caused dephosphorylation (and hence, the activation) of glycogen synthase kinase-3beta (GSK-3beta) with a decline of beta-catenin protein. Accordingly, DHA treatment also decreased the beta-catenin-mediated T cell factor/lymphoid enhancer factor (TCF/LEF) reporter activity, and inhibited the expression of c-Met, a beta-catenin-controlled downstream gene implicated in cholangiocarcinogenesis. The GSK-3beta inhibitor, SB216763, partially prevented DHA-induced reduction of beta-catenin protein and TCF/LEF reporter activity, and restored cell growth, suggesting the involvement of GSK-3beta dephosphorylation in omega 3-PUFA-induced beta-catenin degradation. In parallel, DHA treatment also induced the formation of the beta-catenin/Axin/GSK-3beta binding complex, further leading to beta-catenin degradation. Moreover, DHA inhibited the expression of cyclooxygenase-2 (COX-2) and enhanced the expression of 15-hydroxyprostaglandin dehydrogenase, a physiologic COX-2 antagonist, in human cholangiocarcinoma cells. These findings suggest that omega 3-PUFAs block cholangiocarcinoma cell growth at least in part through inhibition of Wnt/beta-catenin and COX-2 signaling pathways. Thus, utilization of omega 3-PUFAs may represent an effective and safe therapeutic approach for the chemoprevention and treatment of human cholangiocarcinoma.