Gut microbiota regulates the ALK5/NOX1 axis by altering glutamine metabolism to inhibit ferroptosis of intrahepatic cholangiocarcinoma cells.

Intrahepatic cholangiocarcinoma (ICC) is a kind of hepatobiliary tumor that is increasing in incidence and mortality. The gut microbiota plays a role in the onset and progression of cancer, however, the specific mechanism by which the gut microbiota acts on ICC remains unclear. In this study, feces and plasma from healthy controls and ICC patients were collected for 16S rRNA sequencing or metabolomics analysis. Gut microbiota analysis showed that gut microbiota abundance and biodiversity were altered in ICC patients compared with controls. Plasma metabolism analysis showed that the metabolite glutamine content of the ICC patient was significantly higher than that of the controls. KEGG pathway analysis showed that glutamine plays a vital role in ICC. In addition, the use of antibiotics in ICC animals further confirmed that changes in gut microbiota affect changes in glutamine. Further experiments showed that supplementation with glutamine inhibited ferroptosis and downregulated ALK5 and NOX1 expression in HuCCT1 cells. ALK5 overexpression or NOX1 overexpression increased NOX1, p53, PTGS2, ACSL4, LPCAT3, ROS, MDA and Fe2+ and decreased FTH1, SLC7A11 and GSH. Knockdown of NOX1 suppressed FIN56-induced ferroptosis. In vivo, supplementation with glutamine promoted tumor growth. Overexpression of ALK5 repressed tumor growth and induced ferroptosis in nude mice, which could be reversed by the addition of glutamine. Our results suggested that the gut microbiota altered glutamine metabolism to inhibit ferroptosis in ICC by regulating the ALK5/NOX1 axis.

Pharmacokinetic Analysis of Prognostic Factors in Patients With Advanced-Stage Intrahepatic Cholangiocarcinoma Following the Administration of Capsule Formulation of the Standardized Extract of Atractylodes lancea (Thunb) DC.

BACKGROUND: A statistical model is essential in determining the appropriate predictive indicators for therapies in many types of cancers. Predictors have been compared favorably to the traditional systems for many cancers. Thus, this study has been proposed as a new standard approach. A recent study on the clinical efficacy of Atractylodes lancea (Thunb) DC. (AL) revealed the higher clinical benefits in patients with advanced-stage intrahepatic cholangiocarcinoma (ICC) treated with AL compared with standard supportive care. We investigated the relationships between clinical efficacy and pharmacokinetic parameters of serum bioactivity of AL and its active constituent atractylodin and determined therapeutic ranges. METHODS: Group 1 of advanced-stage ICC patients received daily doses of 1000 mg of standardized extract of the capsule formulation of AL (CMC-AL) for 90 days. Group 2 received daily doses of 1000 mg of CMC-AL for 14 days, followed by 1500 mg for 14 days, and 2000 mg for 62 days. Group 3 (control group) received palliative care. Cox proportional hazard model and Receiver Operating Characteristic (ROC) were applied to determine the cut-off values of AUC0-inf, Cmax, and Cavg associated with therapeutic outcomes. Number needed to treat (NNT) and relative risk (RR) were also applied to determine potential predictors. RESULTS: The AUC0-inf of total AL bioactivity of >96.71 µg hour/ml was identified as a promising predictor of disease prognosis, that is, progression-free survival (PFS) and disease control rate (DCR). Cmax of total AL bioactivity of >21.42 was identified as a predictor of the prognosis of survival. The therapeutic range of total AL bioactivity for PFS and DCR is 14.48 to 65.8 µg/ml, and for overall survival is 10.97 to 65.8 µg/ml. Conclusions: The predictors of ICC disease prognosis were established based on the pharmacokinetics of total AL bioactivity. The information could be exploited to improve the clinical efficacy of AL in patients with advanced-stage ICC. These predictors will be validated in a phase 2B clinical study. TRIAL REGISTRATION: TCTR20210129007 (TCTR: www.clinicaltrials.in.th).

The inhibition of ß-catenin activity by luteolin isolated from Paulownia flowers leads to growth arrest and apoptosis in cholangiocarcinoma.

To develop an inhibitor targeting the Wnt/ß-catenin signaling pathway, flavonoid monomer that can interact with ß-catenin was isolated from Paulownia flowers. Luteolin may form stable hydrogen bonds with ß-catenin by molecular docking. Fluorescence quenching analysis determined the physical interaction between luteolin and ß-catenin. The binding of luteolin to ß-catenin caused a loss of α-helical structure and induced a conformational change through circular dichroism spectroscopy. Luteolin inhibits the activity of the Wnt signaling, causing cholangiocarcinoma (CCA) cell cycle arrest in the G2/M phase, leading to cell apoptosis and inhibition of cell migration. In addition, transcriptome and proteomics analysis showed that the differentially expressed proteins were significantly enriched in the Wnt/ß-catenin pathway. ß-catenin protein in the nucleus was significantly decreased, while C-Myc and cyclin D1 in the CCA cells were significantly decreased after luteolin treatment. Additionally, activation of the Wnt/ß-catenin signaling reversed the inhibitory effect of luteolin on the migration of CCA cells. Therefore, luteolin can directly interact with ß-catenin and act as an inhibitor of ß-catenin, inhibiting proliferation and reducing the migration ability of CCA cells by inhibiting the Wnt/ß-catenin pathway. This study provides a scientific basis for the development of Wnt/ß-catenin pathway inhibitors and the prevention and treatment of CCA.

Induction of apoptotic cell death of cholangiocarcinoma cells by tiliacorinine from Tiliacora triandra: A mechanistic insight.

BACKGROUND: Cholangiocarcinoma (CCA) exhibits poor response to the present chemotherapeutic agents and frequently develops drug resistance. Finding novel anticancer drugs might enhance patient outcomes. Tiliacorinine, a bisbenzylisoquinoline alkaloid from the Thai medicinal plant Tiliacora triandra, effectively induced apoptosis of human CCA cell lines and inhibited tumor growth in mice. Here, we elucidate further the molecular mechanisms underlining the cytotoxicity of tiliacorinine and its implication in overcoming gemcitabine-resistance of CCA cells. METHODS: Cytotoxicity of tiliacorinine against CCA cell lines was assessed using MTT assay. The molecular signaling was determined using Western blot analysis. Molecular docking simulations were applied to predict the binding affinity and orientation of tiliacorinine to the possible binding site(s) of the target proteins. RESULTS: Tiliacorinine induced apoptotic cell death of CCA cells in a dose- and time-dependent manner. Tiliacorinine significantly suppressed the expression of anti-apoptotic proteins, Bcl-xL and XIAP; activated apoptotic machinery proteins, caspase-3, caspase-9, and PARP; and decreased the levels of pAkt and pSTAT3. EGF/EGFR activation model and molecular docking simulations revealed EGFR, Akt, and STAT3 as potent targets of tiliacorinine. Molecular docking simulations indicated a strong binding affinity of tiliacorinine to the ATP-binding pockets of EGFR, PI3K, Akt, JAK2, and SH2 domain of STAT3. Tiliacorinine could synergize with gemcitabine and restore the cytotoxicity of gemcitabine against gemcitabine-resistant CCA cells. CONCLUSION: Tiliacorinine effectively induced apoptosis via binding and blocking the actions of EGFR, Akt, and STAT3. GENERAL SIGNIFICANCE: Tiliacorinine is a novel multi-kinase inhibitor and possibly a potent anti-cancer agent, in cancers with high activation of EGFR.

A Retrospective Analysis of Biliary Tract Cancer Patients Presented to the Molecular Tumor Board at the Comprehensive Cancer Center Munich.

BACKGROUND AND OBJECTIVE: With the rising importance of precision oncology in biliary tract cancer (BTC), the aim of this retrospective single-center analysis was to describe the clinical and molecular characteristics of patients with BTC who underwent comprehensive genomic profiling (CGP) and were discussed in the CCCMunichLMU molecular tumor board (MTB). PATIENTS AND METHODS: In this single-center observational study, we included BTC patients with intrahepatic cholangiocarcinoma (iCCA), extrahepatic CCA (eCCA), and gallbladder cancer (GB), who had been discussed in the institutional MTB from May 29, 2017, to July 25, 2022. Patients were followed up until 31 January 2023. Data were retrospectively collected by review of medical charts, and MTB recommendation. RESULTS: In total, 153 cases were registered to the MTB with a median follow-up of 15 months. Testing was successful in 81.7% of the patients. CGP detected targetable alterations in 35.3% of our BTC patients (most commonly ARID1A/ERBB2/IDH1/PIK3CA/BRAF-mutations and FGFR2-fusions). Recommendations for molecularly guided therapy were given in 46.4%. Of those, treatment implementation of targeted therapy followed in 19.4%. In patients receiving the recommended treatment, response rate was 57% and median overall survival was 19 months (vs 8 months in the untreated cohort). The progression-free survival ratio of 1.45 suggest a clinical benefit of molecularly guided treatment. CONCLUSIONS: In line with previous work, our series demonstrates feasibility and clinical utility of comprehensive genomic profiling in BTC patients. With the growing number of targeted agents with clinical activity in BTC, CGP should become standard of care in the management of this group of patients.

Preclinical studies of toxicity and anti-cholangiocarcinoma activity of the standardized capsule formulation of Atractylodes lancea (Thunb.) DC.

BACKGROUND: Cholangiocarcinoma (CCA), the adenocarcinoma of the biliary duct, is commonly reported in Asia, with the highest incidence in northeastern Thailand. Chemotherapy of CCA has been limited by the lack of effective chemotherapeutic drugs. A series of previous in vitro and in vivo studies support further research and development of Atractylodes lancea (Thunb.) DC. (AL) as a potential candidate for treating CCA as a crude ethanolic extract. In the present study, we evaluated the toxicity and anti-CCA activity of the CMC (Chemistry, Manufacturing, and Control) capsule formulation of the ethanolic rhizome extract of AL (CMC-AL) in animals. METHODS: Major steps included acute, subchronic and chronic toxicity testing in Wistar rats and anti-CCA activity in a CCA-xenografted nude mouse model. The safety of CMC-AL was determined based on the maximum tolerated dose (MTD) and no-observed-adverse-effect level (NOAEL) according to the OECD guideline. The anti-CCA activity of CMC-AL in nude mice was evaluated after transplantation of CL-6 cells to evaluate inhibitory effects on tumor size progression and metastasis and survival time prolongation. Safety assessments included hematology, biochemistry parameters and histopathological examination. Lung metastasis was investigated using VEGF ELISA kit. RESULTS: All evaluations confirmed satisfactory pharmaceutical properties of oral formulation and safety profile of the CMC-AL with no overt toxicity up to the MTD and NOAEL of 5,000 and 3,000 mg/kg body weight, respectively. CMC-AL exhibited potent anti-CCA efficacy with regard to inhibitory activity on tumor progression and lung metastasis. CONCLUSIONS: CMC-AL is safe and should be further investigated in a clinical trial as a potential therapy for CCA patients.

Does Adjuvant Therapy Benefit Low-Risk Resectable Cholangiocarcinoma? An Evaluation of the NCCN Guidelines.

BACKGROUND: The National Comprehensive Cancer Network (NCCN) guidelines recommend adjuvant therapy for patients with resectable cholangiocarcinoma (CCA). The trends in utilization and receipt of adjuvant therapy and its association with overall survival have not been well studied among patients with low-risk CCA. METHODS: Patients who received systemic chemotherapy for low-risk CCA after surgical resection (2010-2017) were identified in the National Cancer Database. Low-risk CCA was defined according to NCCN guidelines as patients with R0 margins and negative regional lymph nodes. Multivariable analysis was performed to assess predictors of NCCN guideline concordance and its association with overall survival. RESULTS: Among 4519 patients who underwent resection for low-risk CCA, 55.5% (n = 2510) had intrahepatic, 15.0% (n = 680) had perihilar, and 29.4% (n = 1329) had distal cholangiocarcinoma. Adherence to NCCN guidelines increased from 27.7% in 2010 to 41.6% in 2017 (ptrend < 0.001) for low-risk CCA. On multivariable analysis, receipt of NCCN guideline-concordant care was associated with a nearly 15% decrease in mortality hazards (HR 0.86, 95%CI 0.78-0.95, [Formula: see text]). Increased distance travelled (Ref < 12.5 miles, 50-249 miles: OR 0.55, 95%CI 0.49-0.69; ≥ 250 miles: OR 0.41, 95%CI 0.25-0.6), and care in the South (OR 0.78, 95%CI 0.64-0.95) or Midwest (OR 0.66, 95%CI 0.53-0.81) of the United States versus the Northeast was associated with not receiving guideline-concordant care. CONCLUSION: Adherence to evidence-based NCCN guidelines was associated with improved survival among low-risk CCA patients. Geographical disparities in the receipt of NCCN guideline-concordant care exist and may influence long-term outcomes among CCA patients.

Label-Free Imaging Analysis of Patient-Derived Cholangiocarcinoma Organoids after Sorafenib Treatment.

Monitoring tumor growth dynamics is crucial for understanding cancer. To establish an in vitro method for the continuous assessment of patient-specific tumor growth, tumor organoids were generated from patients with intrahepatic CCA (iCCA). Organoid growth was monitored for 48 h by label-free live brightfield imaging. Growth kinetics were calculated and validated by MTS assay as well as immunohistochemistry of Ki67 to determine proliferation rates. We exposed iCCA organoids (iCCAOs) and non-tumor intrahepatic cholangiocyte organoids (ICOs) to sub-therapeutic concentrations of sorafenib. Monitoring the expansion rate of iCCAOs and ICOs revealed that iCCAO growth was inhibited by sorafenib in a time- and dose-dependent fashion, while ICOs were unaffected. Quantification of the proliferation marker Ki67 confirmed inhibition of iCCAO growth by roughly 50% after 48 h of treatment with 4 µM sorafenib. We established a robust analysis pipeline combining brightfield microscopy and a straightforward image processing approach for the label-free growth monitoring of patient-derived iCCAOs. Combined with bioanalytical validation, this approach is suitable for a fast and efficient high-throughput drug screening in tumor organoids to develop patient-specific systemic treatment options.

Differential in vitro effects of targeted therapeutics in primary human liver cancer: importance for combined liver cancer.

The incidence of primary liver tumors, hepatocellular carcinoma (HCC), intrahepatic cholangiocellular carcinoma (ICC), and combined HCC/ICC (cHCC/CC) is increasing. For ICC, targeted therapy exists only for a small subpopulation of patients, while for HCC, Sorafenib and Lenvatinib are in use. Diagnosis of cHCC/CC is a great challenge and its incidence is underestimated, bearing the risk of unintended non-treatment of ICC. Here, we investigated effects of targeted inhibitors on human ICC cell lines (HUH28, RBE, SSP25), in comparison to extrahepatic (E)CC lines (EGI1, CCC5, TFK1), and HCC/hepatoblastoma cell lines (HEP3B, HUH7, HEPG2). Cells were challenged with: AKT inhibitor MK-2206; multikinase inhibitors Sorafenib, Lenvatinib and Dasatinib; PI3-kinase inhibitors BKM-120, Wortmannin, LY294002, and CAL-101; and mTOR inhibitor Rapamycin. Dosage of the substances was based on the large number of published data of recent years. Proliferation was analyzed daily for four days. All cell lines were highly responsive to MK-2206. Thereby, MK-2206 reduced expression of phospho(p)-AKT in all ICC, ECC, and HCC lines, which mostly corresponded to reduction of p-mTOR, whereas p-ERK1/2 was upregulated in many cases. Lenvatinib showed inhibitory effects on the two HCC cell lines, but not on HEPG2, ICCs and ECCs. Sorafenib inhibited proliferation of all cells, except the ECC line CCC5. However, at reduced dosage, we observed increased cell numbers in some ICC experiments. Dasatinib was highly effective especially in ICC cell lines. Inhibitory effects were observed with all four PI3-kinase inhibitors. However, cell type-specific differences were also evident here. Rapamycin was most effective in the two HCC cell lines. Our studies show that the nine inhibitors differentially target ICC, ECC, and HCC/hepatoblastoma lines. Caution should be taken with Lenvatinib and Sorafenib administration in patients with cHCC/CC as the drugs may have no effects on, or might even stimulate, ICC.

Anti-metastatic Potential of Natural Triterpenoid Cucurbitacin B Against Cholangiocarcinoma Cells by Targeting Src Protein.

Owing to the crucial role of Src in cancer metastasis, interruption of Src and its signaling has been considered a promising strategy for cancer metastasis treatment. Cucurbitacin B, a dietary triterpenoid, has been shown to possess anti-proliferative and apoptosis-inducing activities in cholangiocarcinoma (CCA) cells via suppressing the activation of FAK which is a main downstream Src effector. We hypothesized that cucurbitacin B might act as a Src suppressant which conferring anti-metastasis effect against CCA cells. To investigate this, the role of Src in regulating metastasis behavior of CCA cells and the effect of cucurbitacin B on Src-mediated metastatic phenotype of these cells were determined. The results showed that activation of Src significantly enhanced the migratory and invasive abilities of CCA cells. Molecular analysis revealed that Src-facilitated metastasis behavior of CCA cells occurred by modifying expression of a wide range of metastasis-related genes in the cells. Consistent with gene expression results, activation of Src significantly induced the protein expression of 2 important metastasis-associated molecules, MMP-9 and VEGF. Cucurbitacin B markedly suppressed activation of Src and its key effector, FAK. As a consequence, the alteration of expression profiles of metastasis-associated genes induced by Src activator in CCA cells was diminished by cucurbitacin B treatment. The compound also down-regulated Src-induced expression of MMP-9 and VEGF proteins in the cells. Moreover, molecular docking analysis revealed that cucurbitacin B could interact with Src kinase domain and possibly restrain the kinase from being activated by hindering the ATP binding. In conclusion, cucurbitacin B exhibited anti-metastatic property in CCA cells via negatively influencing Src and Src-related oncogenic signaling. This compound may therefore be a potential therapeutic drug for further development as an anti-Src agent for treatment of metastatic CCA.

Apoptotic and Anti-metastatic Effects of Atractylodes lancea (Thunb.) DC. in a Hamster Model of Cholangiocarcinoma.

OBJECTIVES: Cholangiocarcinoma (CCA) is a highly aggressive tumor with a greater risk of distant metastasis. The promising anti-CCA activity and safety profile of Atractylodes lancea (AL) have previously been reported in a series of in vitro, in vivo and clinical studies. The present study investigated the effect of AL extract on apoptosis and metastasis signaling pathways in the Opisthorchis viverrini/dimethylnitrosamine (OV/DMN)-induced CCA hamster model. MATERIALS AND METHODS: Hamster liver tissues were obtained from the four groups (n = 5 per group), i.e., (i) 5-FU treated CCA (40 µg/mL); (ii) CCA; (iii) AL-treated CCA (5,000 mg/kg), and (iv) normal hamsters. Total RNA was isolated, and the expression levels of apoptosis-related and metastasis-related genes were determined by qRT-PCR analysis. RESULTS: The expression levels of p16, caspase-3, caspase-8, caspase-9, Apaf-1, p53 and Eef1a1 were downregulated, while that of the remaining genes were upregulated in CCA hamsters compared with normal hamsters. AL treatment increased the expression of p16, caspase-9, caspase-3, Apaf-1, p53 and E-cadherin and decreased the expression of cyclin D1, cdk4, Bax, Akt/PKB, Bcl-2, Mfge-8, Lass4, S100A6, TGF-ß, Smad-2, Smad-3, Smad-4, MMP-9, and N-cadherin. The expression of Eef1a1 was unchanged. CONCLUSION: The anti-CCA activity of AL in OV/DMN-induced CCA hamsters could be due to the induction of cell cycle arrest at the G1 phase and activation of the apoptosis pathway, resulting in cancer cell death. The activation of the apoptosis pathway mainly involved the intrinsic pathway (activation of caspase-3 and caspase-9 through p53 and Mfge-8 modulation and downregulation of anti-apoptotic genes Akt and Bcl-2). In addition, AL could also inhibit the canonical TGF-ß signaling pathway, MMP-9 and N-cadherin to suppress tumor metastasis.

Patient-derived functional organoids as a personalized approach for drug screening against hepatobiliary cancers.

Patient-derived organoids (PDOs) established from hepatobiliary cancers are seen as valuable models of the cancer of origin. More precisely, PDOs have the ability to retain the original cancer genetic, epigenetic and phenotypic features. By extension, hepatobiliary cancer PDOs have the potential to (1) increase our understanding of cancer biology; (2) allow high-throughput drug screening for more efficient identification and testing of small molecule therapeutics, and (3) permit the design of personalized drug choice approaches for patients with liver cancer. Here, we review general principles for PDO establishment from hepatocellular carcinoma and cholangiocarcinoma, their utilization in drug screening strategies, and last, the establishment of complex PDOs to include tumor stroma. We conclude that PDOs represent a promising and important development in investigating interaction between liver cancer cell types and their microenvironment, as well as for positioning PDOs for high throughput drug screening for hepatobiliary cancers, and that further work is now needed to fully realize their potential.

YAP1 activation and Hippo pathway signaling in the pathogenesis and treatment of intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA), the second most common primary liver cancer, is a highly lethal epithelial cell malignancy exhibiting features of cholangiocyte differentiation. iCCAs can potentially develop from multiple cell types of origin within liver, including immature or mature cholangiocytes, hepatic stem cells/progenitor cells, and from transdifferentiation of hepatocytes. Understanding the molecular mechanisms and genetic drivers that diversely drive specific cell lineage pathways leading to iCCA has important biological and clinical implications. In this context, activation of the YAP1-TEAD dependent transcription, driven by Hippo-dependent or -independent diverse mechanisms that lead to the stabilization of YAP1 is crucially important to biliary fate commitment in hepatobiliary cancer. In preclinical models, YAP1 activation in hepatocytes or cholangiocytes is sufficient to drive their malignant transformation into iCCA. Moreover, nuclear YAP1/TAZ is highly prevalent in human iCCA irrespective of the varied etiology, and significantly correlates with poor prognosis in iCCA patients. Based on the ubiquitous expression and diverse physiologic roles for YAP1/TAZ in the liver, recent studies have further revealed distinct functions of active YAP1/TAZ in regulating tumor metabolism, as well as the tumor immune microenvironment. In the current review, we discuss our current understanding of the various roles of the Hippo-YAP1 signaling in iCCA pathogenesis, with a specific focus on the roles played by the Hippo-YAP1 pathway in modulating biliary commitment and oncogenicity, iCCA metabolism, and immune microenvironment. We also discuss the therapeutic potential of targeting the YAP1/TAZ-TEAD transcriptional machinery in iCCA, its current limitations, and what future studies are needed to facilitate clinical translation.

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.

Epidermal growth factor receptor as a potential target of momordin Ic to promote apoptosis of cholangiocarcinoma cells.

OBJECTIVES: Strategies that induce apoptosis of malignant cells are recognized as effective cancer treatments. This study evaluated the apoptosis-inducing ability of momordin Ic against cholangiocarcinoma (CCA) cells and the respective underlying mechanisms. METHODS: Quantification of apoptotic cells was performed using Annexin V/7-AAD double dye staining followed by flow cytometry. The effect of momordin Ic on the expression of epidermal growth factor receptor (EGFR) and its downstream signalling molecules was determined via Western blot analysis. The RT2 Profiler PCR Array was used to determine the expression of cell death-associated genes. Expression levels of apoptosis-related proteins were examined using an apoptosis antibody array. KEY FINDINGS: Momordin Ic potently limited the ability of CCA cells to thrive by promoting apoptotic cell death. This apoptosis-inducing activity was accompanied with suppression of expression of EGFR, p-EGFR, c-Myc and other downstream EGFR signalling-related molecules. Additional molecular analyses demonstrated that momordin Ic modified the expression profile of cell death-associated genes in CCA cells. Moreover, significant upregulation of apoptosis-activating proteins and downregulation of apoptosis-inhibiting protein were also observed after exposure to momordin Ic. CONCLUSIONS: These results suggest that momordin Ic has a potential therapeutic opportunity for CCA treatment by acting as an EGFR suppressant.

Clinical application of indocyanine green fluorescence imaging in laparoscopic lymph node dissection for intrahepatic cholangiocarcinoma: A pilot study (with video).

BACKGROUND: Intrahepatic cholangiocarcinoma is a highly lethal malignancy characterized by lymph node metastasis. This study aimed to evaluate the efficacy of indocyanine green fluorescence for visualization of lymphatic drainage and to assess its clinical application during laparoscopic lymph node dissection for intrahepatic cholangiocarcinoma. METHODS: All patients with intrahepatic cholangiocarcinoma who underwent laparoscopic left hepatectomy and lymph node dissection between October 2018 and January 2021 were reviewed. The patients were assigned to the indocyanine green group or non-intrahepatic cholangiocarcinoma group based on the staining technique used. RESULTS: Of 38 patients with left hemiliver intrahepatic cholangiocarcinoma, 20 underwent intrahepatic cholangiocarcinoma tracer-guided laparoscopic radical left hepatectomy; 12 procedures were successful (indocyanine green group). During the same period, 18 patients were treated with traditional laparoscopic resection (control group). Their intraoperative factors were comparable and there were no differences in the incidence or severity of their postoperative complications 30 days after surgery (P > .05). In the indocyanine green group, more lymph nodes were harvested (mean [range]: 7.0 [6.0-8.0] vs 3.5 [3.0-5.0], P < .001) and the proportion of confirmed pathologic lymph nodes was higher (75.0%, 66.7%-87.5% vs 40%, 33.3%-50.0%, P < .001). ICG staining was observed in all (12/12, 100%) patients in the intrahepatic cholangiocarcinoma group at stations 8 and 12, and 9 (9/12, 75%) and 10 (11/12, 91.7%) patients at Stations 13 and 7, respectively. CONCLUSION: The indocyanine green fluorescence imaging system is feasible, safe, and effective for tracing lymph nodes. It can be used to identify regional lymphatic drainage patterns and help define the scope of lymph node dissection in patients with intrahepatic cholangiocarcinoma.

Ethnopharmacological nexus between the traditional Thai medicine theory and biologically based cancer treatment.

ETHNOPHARMACOLOGICAL RELEVANCE: The two major theories utilized for diagnosis and treatment in Traditional Thai Medicine (TTM) are the Four Element Theory and the Herbal Flavor Theory. A TTM "Poh-Pu" Remedy has been effectively utilized in Thailand for cancer therapy for centuries. AIMS OF STUDY: To investigate anti-inflammatory activity and liver cancer cytotoxicity of Poh-Pu remedy. To determine relationships between the TTM Herbal Flavor theory and the Four Element theory and total flavonoid content and biological activities of Poh-Pu Remedy plant extracts. MATERIALS AND METHODS: Each plant ingredient was macerated with 95% ethanol. The extracts were investigated for cytotoxic activity against liver cancer using a sulforhodamine B assay, and anti-inflammatory activity was evaluated by inhibition of nitric oxide production. The total flavonoid content was determined by an aluminum chloride colorimetric assay. The relationships between the TTM theories, total flavonoid content, and biological activities were evaluated by correlation and cluster analysis. RESULTS: Mammea siamensis exerted potent cytotoxicity against hepatocellular carcinoma (HepG2) cell lines with an IC50 of 3.15 ± 0.16 µg/mL and low cytotoxicity to the non-cancerous cells (HaCat) with an IC50 33.39 ± 0.40 µg/mL (Selective index (SI) = 10.6). Tiliacora triandra was selectively cytotoxic to cholangiocarcinama (KKU-M156) cells with an IC50 of 12.65 ± 0.92 µg/mL (SI = 6.4). Curcuma comosa was the most potent anti-inflammatory inhibitor of nitric oxide production with an IC50 of 2.75 ± 0.34 µg/mL. Campomanesia aromatica exhibited the highest total flavonoid content of 259.7 ± 3.21 mg quercetin equivalent/g. Pungent plants were most prevalent in the TTM remedy. CONCLUSION: Pungent, fragrant, bitter and nauseating plants utilized in TTM cancer remedy were successfully investigated and identified several lead plants and components with cytotoxic and antiinflammatory activity that require further study. The TTM wind element theory appeared to be aligned with cancer-related activity. Biological activity results of taste from herbs related with The TTM Herbal Flavor theory. The extra-oral locations of flavor receptors are a promising target for biological activity of TTM which require further scrutiny and identified several lead plants and components with cytotoxic and antiinflammatory activities that also require further study.

Cordycepin Sensitizes Cholangiocarcinoma Cells to Be Killed by Natural Killer-92 (NK-92) Cells.

Immunotherapy harnessing immune functions is a promising strategy for cancer treatment. Tumor sensitization is one approach to enhance tumor cell susceptibility to immune cell cytotoxicity that can be used in combination with immunotherapy to achieve therapeutic efficiency. Cordycepin, a bioactive compound that can be extracted from some Cordyceps spp. has been reported to effectively inhibit tumor growth, however, the mechanism of its tumor sensitization activity that enhances immune cell cytotoxicity is unknown. In the present study, we investigated the potency of cordycepin to sensitize a lethal cancer, cholangiocarcinoma (CCA), to natural killer (NK) cells. Treatment with cordycepin prior to and during co-culturing with NK-92 cells significantly increased cell death of KKU-213A as compared to solitary cordycepin or NK treatment. Moreover, sensitization activity was also observed in the combination of NK-92 cells and Cordyceps militaris extract that contained cordycepin as a major component. The cordycepin treatment remarkably caused an increase in TRAIL receptor (DR4 and DR5) expression in KKU-213A, suggesting the possible involvement of TRAIL signaling in KKU-213A sensitization to NK-92 cells. In conclusion, this is the first report on the sensitization activity of cordycepin on CCA cells to NK cytotoxicity, which supports that cordycepin can be further developed as an alternate immunomodulating agent.