Cytotoxic activities and effects of atractylodin and ß-eudesmol on the cell cycle arrest and apoptosis on cholangiocarcinoma cell line.

Cholangiocarcinoma (CCA) is the cancer of bile duct with high mortality rate particularly in Thailand. The clinical efficacy of the standard chemotherapeutics remains unsatisfactory, and therefore, discovery and development of the new alternative drugs with high efficacy and tolerability is needed. The aim of the study was to investigate cytotoxic activity as well as the underlying mechanisms through which atractylodin and ß-eudesmol exert their activities on CCA cell growth inhibition, cell cycle arrest, and cell apoptosis. Effects of the compounds on cell cytotoxicity, cell cycle arrest, and cell apoptosis were analyzed using MTT assay, BD Cycletest™ Plus DNA kit, and FITC Annexin V Apoptosis Detection Kit I, respectively. The cytotoxic activities of both compounds were concentration- and time-dependent. The IC50 [mean (SD)] of atractylodin and ß-eudesmol were 41.66 (2.51) and 39.33 (1.15) µg/ml respectively. Both promoted cell cycle arrest at G1 phase, and induced cell apoptosis through activation of caspase-3/7. The highest activity was observed at 48 h of exposure. Results suggest that these mechanisms are at least in part, explain the cell cytotoxic and anti-CCA activity of atractylodin and ß-eudesmol shown in vitro and in vivo models.

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.

The Proteomics and Metabolomics Analysis for Screening the Molecular Targets of Action of ß-Eudesmol in Cholangiocarcinoma.

OBJECTIVE: ß-eudesmol is the active compound isolated from Atractylodes lancea (Thunb) D.C. The actions of this compound against cholangiocarcinoma (CCA) cells include anti-angiogenesis and anti-cell proliferation and growth. For more understanding of the molecular targets of action of ß-eudesmol, the CCA cells (CL-6) were exposed to ß-eudesmol for 24 and 48 hours. METHODS: Proteins and metabolites from the intra- and extra-cellular components of the CL-6 cells were extracted and identified by LC-MS/MS.  Protein analysis was performed using the Venn diagram (protein grouping), PANTHER (gene ontology), and STITCH software (protein-protein interaction). Metabolite analysis including their interactions with proteins, was performed using MetaboAnalyst software. RESULTS: The analysis showed that the actions of ß-eudesmol were associated with various biological processes particularly apoptosis and cell cycle. These included blood coagulation, wound healing, DNA repair, PI3K-Akt signaling pathway, immune system process, MAPK cascade, urea cycle, purine metabolism, ammonia recycling, and methionine metabolism. CONCLUSION: Possible molecular targets of action of ß-eudesmol against CL-6 for cell apoptosis induction were TNFRSf6, cytochrome C, BAX3, DHCR24, CD29, and ATP.  On the other hand, possible targets for cell cycle arrest induction were CDKN2B, MLF1, TFDP2, CDK11-p110, and nicotinamide.

Proteomics Analysis for Identification of Potential Cell Signaling Pathways and Protein Targets of Actions of Atractylodin and ß-Eudesmol Against Cholangiocarcinoma.

OBJECTIVE: The study aimed to identify potential cell signaling pathways and protein targets of actions of atractylodin and ß-eudesmol in cholangiocarcinoma, the two active compounds isolated from Atracylodes lancea using proteomics approach. METHOD: The cholangiocarcinoma cell line, CL-6, was treated with each compound for 3 and 6 hours, and the proteins from both intra- and extracellular components were extracted. LC-MS/MS was applied following the separation of the extract proteins by SDS-PAGE and digestion with trypsin. Signaling pathways and protein expression were analyzed by MASCOT and STITCH software. RESULTS: A total of 4,323 and 4,318 proteins were identified from intra- and extracellular components, respectively. Six and 4 intracellular proteins were linked with the signaling pathways (apoptosis, cell cycle control, and PI3K-AKT) of atractylodin and ß-eudesmol, respectively. Four and 3 extracellular proteins were linked with the signaling pathways (NF-κB and PI3K-AKT) of atractylodin and ß-eudesmol, respectively. CONCLUSION: In conclusion, a total of 17 proteins associated with four cell signaling pathways that could be potential molecular targets of anticholangiocarcinoma action of atractylodin and ß-eudesmol were identified through the application of proteomics approach.

Screening of Molecular Targets of Action of Atractylodin in Cholangiocarcinoma by Applying Proteomic and Metabolomic Approaches.

Cholangiocarcinoma (CCA) is cancer of the bile duct and the highest incidence of CCA in the world is reported in Thailand. Our previous in vitro and in vivo studies identified Atractylodes lancea (Thunb) D.C. as a promising candidate for CCA treatment. The present study aimed to examine the molecular targets of action of atractylodin, the bioactive compound isolated from A. lancea, in CCA cell line by applying proteomic and metabolomic approaches. Intra- and extracellular proteins and metabolites were identified by LC-MS/MS following exposure of CL-6, the CCA cell line, to atractylodin for 24 and 48 h. Analysis of the protein functions and pathways involved was performed using a Venn diagram, PANTHER, and STITCH software. Analysis of the metabolite functions and pathways involved, including the correlation between proteins and metabolites identified was performed using MetaboAnalyst software. Results suggested the involvement of atractylodin in various cell biology processes. These include the cell cycle, apoptosis, DNA repair, immune response regulation, wound healing, blood vessel development, pyrimidine metabolism, the citrate cycle, purine metabolism, arginine and proline metabolism, glyoxylate and dicarboxylate metabolism, the pentose phosphate pathway, and fatty acid biosynthesis. Therefore, it was proposed that the action of atractylodin may involve the destruction of the DNA of cancer cells, leading to cell cycle arrest and cell apoptosis.

Plasma Peptidome as a Source of Biomarkers for Diagnosis of Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is the bile duct cancer which constitutes one of the important public health problems in Thailand with high mortality rate, especially in the Opisthorchis viverrini (a parasite risk factor for CCA) endemic area of the northeastern region of the country. This study aimed to identify potential biomarkers from the plasma peptidome by CCA patients. Peptides were isolated using 10 kDa cut-off filter column and the flow-through was then used as a peptidome for LC-MS/MS analysis. A total of 209 peptides were obtained. Among these, 15 peptides were concerned with signaling pathways and 12 related to metabolic, regulatory, and biosynthesis of secondary metabolite pathways. Five exclusive peptides were identified as potential biomarkers, i.e. ETS domain-containing transcription factor ERF (P50548), KIAA0220 (Q92617), phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform isoform 1 (P42338), LP2209 (Q6XYC0), and casein kinase II subunit alpha (P19784). Three of these biomarkers are signaling related molecules. A combination of these biomarkers for CCA diagnosis is proposed.

Plasma phosphoproteome and differential plasma phosphoproteins with opisthorchis viverrini-related cholangiocarcinoma.

This study was conducted to investigate the plasma phosphoproteome and differential plasma phosphoproteins in cases of of Opisthorchis viverrini (OV)-related cholangiocarcinoma (CCA). Plasma phosphoproteomes from CCA patients (10) and non-CCA subjects (5 each for healthy subjects and OV infection) were investigated using gel-based and solution-based LC-MS/MS. Phosphoproteins in plasma samples were enriched and analyzed by LC-MS/MS. STRAP, PANTHER, iPath, and MeV programs were applied for the identification of their functions, signaling and metabolic pathways; and for the discrimination of potential biomarkers in CCA patients and non-CCA subjects, respectively. A total of 90 and 60 plasma phosphoproteins were identified by gel-based and solution-based LC-MS/MS, respectively. Most of the phosphoproteins were cytosol proteins which play roles in several cellular processes, signaling pathways, and metabolic pathways (STRAP, PANTHER, and iPath analysis). The absence of serine/arginine repetitive matrix protein 3 (A6NNA2), tubulin tyrosine ligase-like family, member 6, and biorientation of chromosomes in cell division protein 1-like (Q8NFC6) in plasma phosphoprotein were identified as potential biomarkers for the differentiation of healthy subjects from patients with CCA and OV infection. To differentiate CCA from OV infection, the absence of both serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit beta isoform and coiled-coil domain-containing protein 126 precursor (Q96EE4) were then applied. A combination of 5 phosphoproteins may new alternative choices for CCA diagnosis.