Interleukin-6 and Lymphocyte-to-Monocyte Ratio Indices Identify Patients with Intrahepatic Cholangiocarcinoma.

BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (iCCA) is a fatal biliary tract cancer with a dismal prognosis due to ineffective diagnostic tools with limited clinical utility. This study investigated peripheral blood indices and cytokine levels to diagnose iCCA. METHODS: Blood samples were collected from healthy subjects (n = 48) and patients with advanced-stage iCCA (n = 47) during a phase I and then phase II trial, respectively. Serum cytokines were measured using a flow cytometer. The peripheral blood indices were estimated based on laboratory data. Multi-linear regression analysis was applied, followed by a probability transformation. The cut-off value and model accuracy were determined using the receiver operating curve (ROC) and the area under the curve (AUC). RESULTS: The interleukin-6 (IL6) and lymphocyte-to-monocyte ratio (LMR) were potential predictors of iCCA [AUC = 0.91 (0.85-0.97) and 0.81 (0.68-0.93); sensitivity = 0.70 and 0.91; specificity = 0.91 and 0.85, respectively]. Patients with IL6 concentrations higher than 11.635 pg/mL (OR = 23.33, p < 0.001) or LMR lower than 7.2 (OR = 58.08, p < 0.001) are at risk of iCCA development. Patients with IL6 levels higher than 21.83 pg/mL, between 15.95 and 21.83 pg/mL, between 8.8 and 15.94 pg/mL, and lower than 8.8 pg/mL were classified as very high-, high-, intermediate-, and low-risk, respectively. Patients with an LMR between 1 and 3.37, 3.38 and 5.76, 5.77 and 7.18, and higher than 7.18 were classified as very high-, high-, intermediate-, and low-risk, respectively. CONCLUSIONS: LMR is recommended for iCCA screening since the estimation is based on a routine laboratory test, which is available in most hospitals.

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).

Overexpression of microRNA-205-5p promotes cholangiocarcinoma growth by reducing expression of homeodomain-interacting protein kinase 3.

The microRNA miR-205-5p has diverse effects in different malignancies, including cholangiocarcinoma (CCA), but its effects on CCA progression is unclear. Here we investigated the role and function of miR-205-5p in CCA. Three CCA cell lines and human serum samples were found to have much higher expression levels of miR-205-5p than seen in typical cholangiocyte cell lines and healthy controls. Inhibition of miR-205-5p suppressed CCA cell motility, invasion and proliferation of KKU-213B whereby overexpression of miR-205-5p promoted cell proliferation and motility of KKU-100 cells. Bioinformatics tools (miRDB, TargetScan, miRWalk, and GEPIA) all predicted various miR-205-5p targets. Experiments using miR-205-5p inhibitor and mimic indicated that homeodomain-interacting protein kinase 3 (HIPK3) was a potential direct target of miR-205-5p. Overexpression of HIPK3 using HIPK3 plasmid cloning DNA suppressed migration and proliferation of KKU-100 cells. Notably, HIPK3 expression was lower in human CCA tissues than in normal adjacent tissues. High HIPK3 expression was significantly associated with longer survival time of CCA patients. Multivariate regression analysis indicated tissue HIPK3 levels as an independent prognostic factor for CCA patients. These findings indicate that overexpression of miR-205-5p promotes CCA cells proliferation and migration partly via HIPK3-dependent way. Therefore, targeting miR-205-5p may be a potential treatment approach for CCA.

Tumor necrosis factor-α (TNF-α) -308G >a promoter polymorphism (rs1800629) promotes Asians in susceptibility to Plasmodium falciparum severe malaria: A meta-analysis.

The multifactorial pathogenesis of severe malaria is partly attributed to host genes, such as those encoding cytokines involved in complex inflammatory reactions, namely tumor necrosis factor-alpha (TNF-α). However, the relationship between TNF-α -308G >A gene polymorphism (rs1800629) and the severity of Plasmodium falciparum (P. falciparum) malaria remains unclear, which prompts a meta-analysis to obtain more precise estimates. The present meta-analysis aimed to better understand this correlation and provide insight into its association in populations with different ethnicities. Literature search outcomes included eight eligible articles in which TNF-α -308G >A polymorphism was determined in uncomplicated malaria (UM) and severe malaria (SM) of P. falciparum as represented in the case and control groups. Pooled odds ratios (ORs) and 95% confidence intervals (95% CIs) were estimated in standard homozygous, recessive, dominant, and codominant genetic models. Subgroup analysis was based on ethnicity, i.e., Africans and Asians. The analyses included overall and the modified outcomes; the latter was obtained without the studies that deviated from the Hardy-Weinberg Equilibrium. The significant data also underwent sensitivity treatment but not publication bias tests because the number of studies was less than ten. Interaction tests were applied to differential outcomes between the subgroups. Overall and HWE-compliant analyses showed no significant association between the TNF-α -308G >A polymorphism and susceptibility to P. falciparum SM (ORs = 1.10-1.52, 95%CIs = 0.68-2.79; Pa = 0.24-0.68). Stratification by ethnicity revealed that two significant associations were found only in the Asians favoring SM for dominant (OR = 1.95, 95% CI = 1.06-3.61, Pa = 0.03) and codominant (OR = 1.83, 95% CI = 1.15-2.92, Pa = 0.01) under the random-effects model, but not among the African populations. The two significant Asian associations were improved with a test of interaction with P-value of0.02-0.03. The significant core outcomes were robust. Results of the meta-analysis suggest that TNF-α -308G >A polymorphism might affect the risk of P. falciparum SM, particularly in individuals of Asian descent. This supports ethnicity as one of the dependent factors of the TNF-α -308G >A association with the clinical severity of malaria. Further large and well-designed genetic studies are needed to confirm this conclusion.

The roles of CYP2C19 and CYP3A4 in the in vitro metabolism of ß-eudesmol in human liver: Reaction phenotyping and enzyme kinetics.

ß-eudesmol is a major bioactive component of Atractylodes lancea (AL). AL has been developed as the capsule formulation of standardized AL extract for treating cholangiocarcinoma (CCA). However, the complex constituents of herbal products increase the risk of adverse drug interactions. ß-eudesmol has demonstrated inhibitory effects on rCYP2C19 and rCYP3A4 in the previous research. This study aimed to identify the cytochrome P450 (CYP) isoforms responsible for the metabolism of ß-eudesmol and determine the enzyme kinetic parameters and the metabolic stability of ß-eudesmol metabolism in the microsomal system. Reaction phenotyping using human recombinant CYPs (rCYPs) and selective chemical inhibitors of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 was performed, and enzyme kinetics and metabolic stability were investigated using human liver microsome (HLM). The results suggest that CYP2C19 and CYP3A4 play significant roles in ß-eudesmol metabolism. The disappearance half-life (t1/2 ) and intrinsic clearance (CLint ) of ß-eudesmol were 17.09 min and 0.20 mL/min·mg protein, respectively. Enzyme kinetic analysis revealed the Michaelis-Menten constant (Km ) and maximum velocity (Vmax ) of 16.76 µM and 3.35 nmol/min·mg protein, respectively. As a component of AL, ß-eudesmol, as a substrate and inhibitor of CYP2C19 and CYP3A4, has a high potential for drug-drug interactions when AL is co-administered with other herbs or conventional medicines.

Antiproliferative and Anti-Inflammatory Activities of Deprungsith Formulation and Its Bioactive Compounds Against Mild Psoriasis and Potential of Metabolic Herb-Drug Interactions.

Psoriasis is an incurable, chronic and auto-immune skin disorder with a global prevalence rate of approximately 2-3%. The study investigated the antipsoriasis activities of Deprungsith formulation and its bioactive components and their potential for inhibitory activities on human cytochrome P450 (CYP450). HaCaT and peripheral blood mononuclear cells (PBMCs) from healthy volunteers (n = 9) and psoriasis patients (n = 10) were exposed to Deprungsith formulation (Thai traditional medicine for psoriasis consisting of 16 plants), ethyl p-methoxycinnamate (EPMC), ligustilide and cyclosporin for 24 and 48 h. The antiproliferative, cell apoptosis and cell cycle arrest activities were evaluated using MTT assay and flow cytometry, respectively. The pro-inflammatory cytokine mRNA expression levels were measured using real-time polymerase chain reaction (RT-PCR). The CYP450 inhibitory effect was investigated using a bioluminescent-based CYP450 assay. Deprungsith formulation and the bioactive compounds inhibited HaCaT cells and PBMCs with weak to moderate potencies. EPMC and ligustilide combination produced an additive effect. Most substances arrested cell transition at sub-G1 and S phases, leading to early and late apoptosis induction. With prolonged exposure (48 h), all test substances decreased PBMCs necrosis. The mRNA expression of all pro-inflammatory cytokines was downregulated. Deprungsith formulation, EPMC, ligustilide and ferulic acid inhibited CYP1A2, CYP2C9, CYP2D6 and CYP3A4 activities with weak to moderate potencies. Deprungsith formulation and bioactive components induced cell apoptosis by inhibiting cell transition at specific cell cycle phases, which was correlated with the mRNA downregulation of interleukin (IL-6, IL-12p19, IL-23) and tumor necrosis factor (TNF-α). There is a low risk of potential adverse drug reactions and toxicity due to CYP450 interaction when Deprungsith formulation is concurrently administered with modern medicines.

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.

Modulatory Effects of Atractylodin and ß-Eudesmol on Human Cytochrome P450 Enzymes: Potential Drug-Drug Interactions.

Atractylodin and ß-eudesmol, the major bioactive compounds in Atractylodes lancea, are promising candidates for anti-cholangiocarcinoma. The inhibitory effects of both compounds on human rCYP1A2, rCYP2C9, rCYP2C19, rCYP2D6 and rCYP3A4 enzymes were investigated using luminogenic CYP450 kits. The modulatory effects were investigated in mouse livers following a daily oral dose of atractylodin or ß-eudesmol at 100 mg/kg body weight for 1, 7, 14, and 21 days. The inhibitory effects of both compounds on all rCYP450s were weak (IC50: 167 to >686 µM). ß-Eudesmol showed the most potent inhibitory effect on rCYP2C19 (IC50 = 172.7 µM) and rCYP3A4 (IC50 = 218.6 µM). Results of the ex vivo study showed that short exposure (1-7 days) of atractylodin and ß-eudesmol resulted in the upregulation of mRNA. Prolonged exposure to the daily oral dose for at least 14 days significantly downregulated the expressions of mRNA and proteins, which correlated with the decrease in the activities of mCYP1A2 and mCYP3A11. Based on the results of the ex vivo study, clinical uses of atractylodin or ß-eudesmol for the treatment of cholangiocarcinoma are of concern for the risk of toxicity due to hCYP3A4 inhibition following chronic dosing, as well as the metabolic interaction with the coadministered drugs that are metabolized by hCYP3A4.

Bioassay for total serum bioactivity of Atractylodes lancea.

The study aimed to establish a bioassay for total bioactivity of Atractylodes lancea (AL) in human serum samples. Inhibition of bacterial growth (Staphylococcus aureus ATCC 25923) was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The calibration curve (0, 0.39, 0.78, 1.56, 3.13, 2.56, and 50 ng/µl) was linear with correlation coefficients >0.990. The limit of quantification (LOQ) was 1.66 mg/ml using 20-ml serum sample. The developed bioassay method meets the standard of the bioanalytical method for determination of serum bioactivity of AL.

Molecular Targets and Signaling Pathways in Cholangiocarcinoma: A Systematic Review.

BACKGROUND: Cholangiocarcinoma (CCA) is the second most frequent hepatobiliary cancer after hepatocellular carcinoma with a poor prognosis and limited treatment options. This study aimed to review existing knowledge on the genetic basis of CCA, molecular targets/signaling pathways involved in the pathogenesis, disease progression and prognosis, including potential targets for targeted therapies of CCA. METHODS: The systematic review was performed in compliance with PRISMA guidelines. A systematic search in PubMed and Science Direct databases was performed using the following keywords: "cholangiocarcinoma", AND "molecular target" AND/OR "signaling pathway", AND/OR "targeted therapy", AND/OR "cancer chemotherapy." The eligibility criteria included: i) full-text articles published in English, ii) articles with in vitro and/or in vivo and/or clinical studies of molecular targets/signaling pathwanys related to CCA pathogenesis/disease progression/prognosis and/or targeted therapy. Seventy-three studies that fulfilled the eligibility criteria were finally included in the final data synthesis. RESULTS: A total of 833 relevant articles published up to April 2022 were identified and 73 sttudies that fulfilled the eligibility criteria were finally included in the analysis. The molecular biomarkers and drugs targeting signalling pathways were reported. Recent research has been focused on targeting the apoptotic and cell proliferation pathways, and in addition, the angiogenesis and metastasis pathway. More effort focused on testing the efficacy of combination therapies against the cancer cell and specifically CCA. The PI3K (Phosphoinositide 3-kinases)/ERK/Akt (AKT serine/threonine kinase 1)/mTOR (mammalian target of rapamycin) signaling pathway and HER2 (Human epidermal growth factor receptor 2) and EGFR (Epidermal Growth Factor Receptor) pathways are the most potential targets for CCA therapy. CONCLUSION: The information obtained could be exploited for further development of diagnostic tools for early diagnosis of CCA, as well as effective CCA-targeted therapies.

Atractylodin and ß-eudesmol from Atractylodes lancea (Thunb.) DC. Inhibit Cholangiocarcinoma Cell Proliferation by Downregulating the Notch Signaling Pathway.

OBJECTIVE: Notch signaling pathway has been reported to be involved in the development and progression of various types of cancer, including cholangiocarcinoma (CCA).  Compounds that modulate this signaling pathway could be promising candidates for CCA treatment and control. The study investigated the antiproliferative activities and modulatory effects of atractylodin and ß-eudesmol, the two bioactive compounds of Atractylodes lancea (Thunb.) DC. , on Notch signaling and upstream molecules (Notch1 and Notch2 receptors, JAG1, mTOR, PI3K, and YAP), and downstream molecules (Snail) in HuCCT-1 (CCA cell line) and OUMS-36T-1 (normal fibroblast cell line). Gemcitabine (standard drug for CCA), and Notch inhibitors (DAPT and zebularine) were included in the experiments as positive control compounds. METHODS: The antiproliferative activity was evaluated using MTT assay.  mRNA and protein expression of Notch signaling molecules were evaluated using real-time PCR and Western blot analysis. RESULTS: Atractylodin and ß-eudesmol moderately inhibited HuCCT-1  cell growth with IC50 (concentration that inhibits cell growth by 50%) of 29.00 ± 6.44 and 16.80 ± 4.41 µg/ml (mean±SD), respectively. The direction and extent of the modulatory effects on mRNA and protein expression in the CCA cell line varied with the signaling molecules. Notch1 receptor was shown to be the most promising target of atractylodin and ß-eudesmol in CCA. The level of gene expression was significantly downregulated (0.042 to 0.195 fold of control) after treating HuCC-T1 cells with both compounds at low and high concentrations. The extent and change in Notch1 gene expression correlated well with protein expression. CONCLUSION: The notch signaling pathway could be a promising target of atractylodin and ß-eudesmol in CCA.  
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The Role of Herbal Medicine in Cholangiocarcinoma Control: A Systematic Review.

The growing incidence of cholangiocarcinoma (bile duct cancer) and limited treatment options stimulate a pressing demand for research and the development of new chemotherapeutics against cholangiocarcinoma. This study aimed to systematically review herbs and herb-derived compounds or herbal formulations that have been investigated for their anti-cholangiocarcinoma potential. Systematic literature searches were conducted in three electronic databases: PubMed, ScienceDirect, and Scopus. One hundred and twenty-three research articles fulfilled the eligibility critera and were included in the analysis (68 herbs, isolated compounds and/or synthetic analogs, 9 herbal formulations, and 119 compounds that are commonly found in several plant species). The most investigated herbs were Atractylodes lancea (Thunb.) DC. (Compositae) and Curcuma longa L. (Zingiberaceae). Only A. lancea (Thunb.) DC. (Compositae) has undergone the full process of nonclinical and clinical development to deliver the final product for clinical use. The extracts of A. lancea (Thunb.) DC. (Compositae), Garcinia hanburyi Hook.f. (Clusiaceae), and Piper nigrum L. (Piperaceae) exhibit antiproliferative activities against human cholangiocarcinoma cells (IC50 < 15 µg/mL). Cucurbitacin B and triptolide are herbal isolated compounds that exhibit the most promising activities (IC50 < 1 µM). A series of experimental studies (in vitro, in vivo, and humans) confirmed the anti-cholangiocarcinoma potential and safety profile of A. lancea (Thunb.) DC. (Compositae) and its active compounds atractylodin and ß-eudesmol, including the capsule pharmaceutical of the standardized A. lancea (Thunb.) DC. (Compositae) extract. Future research should be focused on the full development of the candidate herbs to deliver products that are safe and effective for cholangiocarcinoma control.

Effects of ß-eudesmol and atractylodin on target genes and hormone related to cardiotoxicity, hepatotoxicity, and endocrine disruption in developing zebrafish embryos.

Atractylodes lancea, commonly known as Kod-Kamao in Thai, a traditional medicinal herb, is being developed for clinical use in cholangiocarcinoma. ß-eudesmol and atractylodin are the main active components of this herb which possess most of the pharmacological properties. However, the lack of adequate toxicity data would be a significant hindrance to their further development. The present study investigated the toxic effects of selected concentrations of ß-eudesmol and atractylodin in the heart, liver, and endocrine systems of zebrafish embryos. Study endpoints included changes in the expression of genes related to Na/K-ATPase activity in the heart, fatty acid-binding protein 10a and cytochrome P450 family 1 subfamily A member 1 in the liver, and cortisol levels in the endocrine system. Both compounds produced inhibitory effects on the Na/K-ATPase gene expressions in the heart. Both also triggered the biomarkers of liver toxicity. While ß-eudesmol did not alter the expression of the cytochrome P450 family 1 subfamily A member 1 gene, atractylodin at high concentrations upregulated the gene, suggesting its potential enzyme-inducing activity in this gene. ß-eudesmol, but not atractylodin, showed some stress-reducing properties with suppression of cortisol production.

Atractylodes lancea for cholangiocarcinoma: Modulatory effects on CYP1A2 and CYP3A1 and pharmacokinetics in rats and biodistribution in mice.

Atractylodes lancea (Thunb.) DC. (A. lancea: AL) is a promising candidate for the treatment of cholangiocarcinoma (bile duct cancer). The study investigated (i) the propensity of capsule formulation of the standardized extract of AL (formulated AL) to modulate mRNA and protein expression and activities of CYP1A2 and CYP3A1 in rats after long- and short-term exposure, (ii) the pharmacokinetics of atractylodin (ATD: active constituent) after long-term administration of formulated AL, and (iii) the biodistribution of atractylodin-loaded polylactic-co-glycolic acid (ATD-PLGA-NPs) in mice. To investigate CYP1A2 and CYP3A1 modulatory activities following long-term exposure, rats of both genders received oral doses of the formulated AL at 1,000 (low dose), 3,000 (medium dose), and 5,000 (high dose) mg/kg body weight daily for 12 months. For short-term effects, male rats were orally administered the formulated AL at the dose of 5,000 mg/kg body weight daily for 1, 7, 14 and 21 days. The pharmacokinetic study was conducted in male rats after administration of the formulated AL at the dose of 5,000 mg/kg body weight daily for 9 months. The biodistribution study was conducted in a male mouse receiving ATD-PLGA-NPs at the equivalent dose to ATD of 100 mg/kg body weight. The high dose of formulated AL produced an inducing effect on CYP1A2 but an inhibitory effect on CYP3A1 activities in male rats. The low dose, however, did not inhibit or induce the activities of both enzymes in male and female rats. ATD reached maximum plasma concentration (Cmax) of 359.73 ng/mL at 3 h (tmax). Mean residence time (MRT) and terminal phase elimination half-life (t1/2z) were 3.03 and 0.56 h, respectively. The extent of biodistribution of ATD in mouse livers receiving ATD-PLGA-NPs was 5-fold of that receiving free ATD. Clinical use of low-dose AL should be considered to avoid potential herb-drug interactions after long-term use. ATD-PLGA-NPs is a potential drug delivery system for cholangiocarcinoma treatment.

Potential of Alpha-Mangostin-Loaded PLGA Nanoparticles for Cholangiocarcinoma Treatment.

Alpha-mangostin (AM), a significant component isolated from the pericarp of mangosteen (Garcinia mangostana L.), has been demonstrated as a potential compound for the treatment of cholangiocarcinoma (CCA). Due to its hydrophobic nature, however, its clinical uses may be limited by its low aqueous solubility, poor stability, and low bioavailability. The present study aimed to formulate and characterize the AM-loaded PLGA nanoparticles (AM-PLGA-NPs) and further evaluate the antiproliferative and proapoptotic activities, including the inhibitory activities on CCA cell (CL-6 and HuCCT-1) invasion and migration. The AM-PLGA-NPs were prepared using PLGA MW 7000-17,000 and 38,000-54,000 by the solvent displacement method. The methods used to evaluate these activities included a MTT assay, flow-cytometry, QCM ECMatrix cell migration, and cell invasion assays, respectively. The optimized AM-PLGA-NPs were characterized for physical (particle size and morphology, polydispersity index, and zeta potential) and pharmaceutical (encapsulation efficiency, loading efficiency, and drug release profile) parameters. AM-PLGA-NPs showed relatively potent and selective antiproliferative and proapoptotic activities in both CCA cell lines in a concentration- and time-dependent manner. The results revealed that the PLGA nanoparticles could be a suitable nanocarrier to encapsulate AM for its delivery to the CCA cells.

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.

Alpha-mangostin inhibits viral replication and suppresses nuclear factor kappa B (NF-κB)-mediated inflammation in dengue virus infection.

Severe dengue virus (DENV) infection results from viral replication and dysregulated host immune response, which trigger massive cytokine production/cytokine storm. The result is severe vascular leakage, hemorrhagic diathesis, and organ dysfunction. Subsequent to previously proposing that an ideal drug for treatment of DENV infection should efficiently inhibit both virus production and cytokine storm, we discovered that α-mangostin (α-MG) from the pericarp of the mangosteen fruit could inhibit both DENV infection and cytokine/chemokine production. In this study, we investigated the molecular mechanisms underlying the antiviral and anti-inflammatory effects of α-MG. Time-of-drug-addition and time-of-drug-elimination studies suggested that α-MG inhibits the replication step of the DENV life cycle. α-MG inhibited polymerization activity of RNA-dependent RNA polymerase (RdRp) with IC50 values of 16.50 µM and significantly reduced viral RNA and protein syntheses, and virion production. Antiviral and cytokine/chemokine gene expression profiles of α-MG-treated DENV-2-infected cells were investigated by polymerase chain reaction array. α-MG suppressed the expression of 37 antiviral and cytokine/chemokine genes that relate to the NF-κB signaling pathway. Immunofluorescence and immunoblot analyses revealed that α-MG inhibits NF-κB nuclear translocation in DENV-2-infected cells in association with reduced RANTES, IP-10, TNF-α, and IL-6 production. These results suggest α-MG as a potential treatment for DENV infection.

ß-Eudesmol Inhibits the Migration of Cholangiocarcinoma Cells by Suppressing Epithelial-Mesenchymal Transition via PI3K/AKT and p38MAPK Modulation.

BACKGROUND: Cholangiocarcinoma (CCA) is a highly aggressive tumor with a greater risk of distant metastasis. A drug that prevents CCA development and spread is urgently needed.  In this research, we investigated the effect of ß-eudesmol on the migration and invasion and epithelial-mesenchymal transformation (EMT) of the CCA cell line. MATERIALS AND METHODS: MTT and transwell assays were used to investigate the antiproliferative activity, as well as activity on cell migration and cell invasion. Real-time PCR and western blot analysis were used to investigate the expression of EMT marker genes and proteins. RESULTS: ß-eudesmol was shown to exhibit potent antiproliferative activity (IC50 92.25-185.67 µM) and to significantly reduce CCA cell migration and invasion (27.3-62.7%). At both mRNA and protein levels, it significantly up-regulated the expression of epithelial marker E-cadherin (3-3.4-fold), while down-regulated the expression of mesenchymal markers-vimentin (0.6-0.8-fold) and snail-1 (0.4-0.6-fold). Furthermore, ß-eudesmol inhibited PI3K and AKT phosphorylation (0.5-0.8-fold), while activating p38MAPK activity (1.2-3.6-fold). CONCLUSION: Altogether, the anti-metastatic activity of ß-eudesmol might be due to its suppressive effect on EMT via modulating the PI3K/AKT and p38MAPK signaling cascades.

Emulgels Containing Perilla frutescens Seed Oil, Moringa oleifera Seed Oil, and Mixed Seed Oil: Microemulsion and Safety Assessment.

P. frutescens seed oil and M. oleifera seed oil consist of fatty acids and sterols that are beneficial for skin. Mixing of these oils at 1:1 ratio has shown to increase antioxidant activity of oils. This study aims to formulate emulgels containing microemulsions of P. frutescens seed oil, M. oleifera seed oil, and mixed P. frutescens and M. oleifera seed oils. The chemical constituents of P. frutescens seed oil, M. oleifera seed oil, and mixed seed oil are analyzed by gas chromatography/mass spectrometry (GC/MS). The microemulsions are formulated by a phase titration method and characterized for the droplet size, polydispersity index, and zeta potential value using a dynamic light scattering technique. The physical and chemical stability of the microemulsions are investigated using a rheometer and UV-Visible spectrophotometer, respectively. The safety of microemulsion is evaluated on PBMC and human subjects. Emulgels containing three different types of microemulsion are formulated. The results show that P. frutescens seed oil is mainly composed of alpha-linolenic acid, linoleic acid, and oleic acid, whereas M. oleifera seed oil contains a high proportion of oleic acid. Mixed seed oil contains a comparable amount of alpha-linolenic acid and oleic acid. All types of oils are composed of ß-sitosterol as the major plant sterol. Microemulsions of all types of oils are successfully prepared by using Tween 80 as a surfactant due to the largest transparent region of pseudoternary phase diagram. The size, polydispersity index, and zeta potential values of all types of microemulsion are in the acceptable range upon storage at 30 °C for 1 month. Microemulsions exhibit pseudoplastic flow behavior. The percent of remaining oils in all types of microemulsion is more than 90% after storage at 30 °C for 1 month. Emulgels containing three types of microemulsions exhibit good characteristics and no change in viscosity after storage at 4, 30, and 45 °C for 1 month. The safety results reveal that three types of microemulsion do not induce cytotoxicity to PBMC nor induce skin irritation and allergic reactions. Emulgels containing microemulsions developed in this study can be used to safely deliver P. frutescens seed oil, M. oleifera seed oil, and mixed seed oil to human skin.

Notch signaling in the pathogenesis, progression and identification of potential targets for cholangiocarcinoma (Review).

Cholangiocarcinoma (CCA) is an aggressive type of bile duct cancer that is characterized by a high mortality rate due to its late diagnosis and ineffective treatment. The aim of the present systematic review was to analyze the association between Notch signaling and CCA in terms of its pathogenesis, progression and potential treatment targets. Relevant information was gathered from the PubMed, ScienceDirect and Scopus databases using the search terms 'cholangiocarcinoma' AND 'Notch signaling'. Of the 90 articles identified, 28 fulfilled the eligibility criteria and were included in the analysis. It was concluded that overexpression/upregulation of Notch ligands, such as Jagged1 and Notch receptors (Notch1, Notch2 and Notch3), as well as upregulation of the upstream Notch signaling pathway, promoted CCA development and progression. In addition, downregulation of Notch1 signaling through several possible interventions appears to be a promising strategy for inhibition of CCA development and progression. Therefore, the Notch signaling pathway may be considered as a potential target for CCA control.