Background Atractylodes lancea (Thunb) DC. (AL) and bioactive compounds β-eudesmol and atractylodin have been demonstrated in the in vitro and in vivo studies for their potential clinical use in cholangiocarcinoma. The study was a randomized, double-blinded, placebo-controlled phase I clinical trial to evaluate the immunomodulatory effect of AL in human subjects. Methods The modulatory effects of AL and β-eudesmol and atractylodin on TNFα and IL6 expression in PBMCs were measured using real-time PCR. Blood samples were collected from forty-eight healthy subjects following oral administration of a single or multiple dosing of capsule formulation of the standardized AL extract or placebo. Serum cytokine profiles, lymphocyte subpopulations (B lymphocytes, CD8+ cytotoxic T lymphocytes, CD4+ T-helper lymphocytes, and NK cells), and cytotoxic activity of PBMCs against the cholangiocarcinoma cell line CL-6 were evaluated using cytometric bead array (CBA) with flow cytometry analysis. Results AL extract at almost all concentrations significantly inhibited both TNFα and IL6 expression in Con A-mediated inflammation in PBMCs. β-Eudesmol at all concentrations significantly inhibited only IL6 expression. Atractylodin at the lowest concentration significantly inhibited the expression of both cytokines, while the highest concentration significantly inhibited only IL6 expression. The administration of AL at a single oral dose of 1000 mg appeared to decrease IFNγ and IL10 and increase B cell, while significantly increase NK and CD4+ and CD8+ cells. A trend of increasing (compared with placebo) in the cytotoxic activity of PBMCs at 24 h of dosing was observed. AL at multiple dosing of 1000 mg for 21 days tended to decrease the production of all cytokines, while significantly inhibited IL17A production at 24 h of dosing. In addition, a significant increase in CD4+ and CD8+ cells was observed. A trend of increase in the cytotoxic activity of PBMCs was observed at 24 h but terminated at 48 h of dosing. Conclusions The results confirm the immunomodulatory activity of AL in humans. This activity, in complementary with the direct action of AL on inducing cholangiocarcinoma cell apoptosis, suggests its potential role for CCA control. Trial registration Retrospectively registered on 17 October 2020 [Thai Clinical Trials Registry (TCTR: www.clinicaltrials.in.th) Number TCTR20201020001#].

Objective:The study aimed to investigate the inhibitory effects of AL on the ERK signaling molecules (ERK, p-ERK, cyclin D, and eIF4B) and the growth and proliferation of CCA cells. Materials and methods: The viability of the three CCA cell lines CL-6, HuCCT1, and HuH28 was determined using MTT assay. The effect of Ras/ERK inhibitors on protein expression in the presence of AL extract was investigated. The protein extracted from each CCA cell following exposure to AL and/or Ras/ERK inhibitors were separated on 12.5% SDS-PAGE. The analysis of mRNA expression following 48 and 72 hours of AL exposure in comparison with 0 hours (non-exposed cells) was performed by using RT-PCR. Results: The potency of cytotoxic activity of AL (by MTT assay) was about three times higher than the standard drug 5-fluorouracil. The IC 50 (concentration that inhibits cell growth by 50%) of AL for the CL-6, HuCCT-1 and HuH28 cell lines were 29.77±6.64, 35.45±4.96, and 35.32±6.69 µg/mL (mean+SD), respectively. The cells were exposed to AL extract at the IC 50 for 0, 12, 24, 48, and 72 hours in the absence and presence of Ras/ERK inhibitors (salirasib and XMD8-92). Protein expression was determined by Western blot analysis. The results suggested the lack of significant inhibitory effect of AL on ERK at 48 and 72 hours of exposure in all CCA cell types. On the other hand, a significant inhibitory effect was observed with p-ERK expression in all CCA cell types. Cyclin D was significantly down-regulated at 72 hours of exposure in all cell types with different potencies. The expression of eIF4B was markedly inhibited in HuCCT-1 but slightly inhibited in CL-6 and HuH28 cells. Real-time PCR analysis revealed significant down-regulation of ERK following 72 hours of AL exposure in the HuCCT1 and HuH28, but not CL-6 cell. Conclusion: The ERK signaling cascade and downstream molecules are potential targets of action of AL in CCA.

Objective: To investigate cytotoxic activity of ethyl-p-methoxycinnamate (EPMC) including its effect on p-glycoprotein (multidrug resistance-1: mdr-1 gene) in human cholangiocarcinoma cell. Methods: Cytotoxic activity of EPMC against human cholangiocarcinoma (CL-6), fibroblast (OUMS-36T-1F), and colon cancer (Caco-2) cell lines were assessed using MTT assay. Selectivity index (SI) was determined as the ratio of IC 50 (concentration that inhibits cell growth by 50%) of EPMC in OUMS-36T-1F and that in CL-6 cell. Cell cycle arrest and apoptosis in CL-6 cells were investigated by flow cytometry and fluorescent microscopy. Effect of EPMC on mdr-1 gene expression in CL-6 and Caco-2 was determined by real-time PCR. Results: The median (95% CI) IC 50 values of EPMC in CL-6, OUMS-36T-1F, and Caco-2 were 245.5 (243.1-266.7), 899.60 (855.8-966.3) and 347.0 (340.3-356.9) µg/ml, respectively. The SI value of the compound for the CL-6 cell was 3.70. EPMC at IC 50 inhibited CL-6 cell division and induced apoptosis compared to untreated control. EPMC exposure did not induce mdr-1 gene expression in both CL-6 and Caco-2 cells. Conclusion: The results suggest the potential role of EPMC in cholangiocarcinoma with a low possibility of drug resistance induction.

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