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Background: Failure of treatment with gemcitabine in most cholangiocarcinoma (CCA) patients is due to drug resistance. The therapeutic potential of natural plant secondary compounds with minimal toxicity, such as cannabidiol (CBD), is a promising line of investigation in gemcitabine-resistant CCA. We aim to investigate the effects of CBD on gemcitabine-resistant CCA (KKU-213B GemR ) cells in vitro and in vivo., Materials: In vitro, cell proliferation, colony formation, apoptosis and cell cycle arrest were assessed using MTT assay, clonogenicity assay and flow cytometry. The effect of CBD on ROS production was evaluated using the DCFH-DA fluorescent probe. The mechanism exerted by CBD on ER stress-associated apoptosis was investigated by western blot analysis. A gemcitabine-resistant CCA xenograft model was also used and the expression of PCNA and CHOP were evaluated by immunohistochemical analysis., Results: The IC 50 values of CBD for KKU-213B GemR cells ranged from 19.66 to 21.05 µM. For a non-cancerous immortalized fibroblast cell line, relevant values were 18.29 to 19.21 µM. CBD suppressed colony formation by KKU-213B GemR cells in a dose-dependent manner in the range of 10 to 30 µM. CBD at 30 µM significantly increased apoptosis at early (16.37%) (P = 0.0024) and late (1.8%) stages (P < 0.0001), for a total of 18.17% apoptosis (P = 0.0017), in part by increasing ROS production (P < 0.0001). Multiphase cell cycle arrest significantly increased at G0/G1 with CBD 10 and 20 µM (P = 0.004 and P = 0.017), and at G2/M with CBD 30 µM (P = 0.005). CBD treatment resulted in increased expression of ER stress-associated apoptosis proteins, including p-PERK, BiP, ATF4, CHOP, BAX, and cytochrome c. In xenografted mouse, CBD significantly suppressed tumors at 10 and 40 mg/kg·Bw (P = 0.0007 and P = 0.0278, respectively), which was supported by an increase in CHOP, but a decrease in PCNA expression in tumor tissues (P < 0.0001)., Conclusion: The results suggest that CBD exhibits potent anti-cancer activity against gemcitabine-resistant CCA in vitro and in vivo, in part via ER stress-mediated mechanisms. These results indicate that clinical explorative use of CBD on gemcitabine-resistant CCA patients is warranted., (© 2024. The Author(s).)

Atrazine (ATZ), an herbicide widely distributed on a global scale, possess a potential risk for the development of various cancers upon environmental exposure. However, the effect and molecular mechanism of ATZ in cholangiocarcinoma (CCA), is still unclear. This study aimed to investigate the effect of ATZ on the proliferation and migration of CCA cell in vitro. Immortalized human cholangiocytes (MMNK-1) and three CCA cell lines (KKU-055, KKU-100 and KKU-213B) were treated with 0.01 to 100 μM of ATZ and 17β-estradiol (E2). The results showed that, similar to E2, low doses (0.01 to 1 μM) of ATZ promoted the proliferation of all CCA and MMNK-1 cells. ATZ exposure increased non-genomic G protein-coupled estrogen receptor (GPER) expression in the cell membrane and cytoplasm of KKU-213B and KKU-055 cells via G2/M cell cycle accumulation. This, in turn, promoted the proliferation and migration of CCA cells. ATZ exposure induced the upregulation of GPER and increased expression levels of PI3K, p-PI3K, Akt, p-Akt, NF-κB and PCNA. In contrast, following ATZ treatment, the GPER antagonist G15 significantly downregulated the GPER/PI3K/Akt/NF-κB pathway. These results suggest that ATZ promotes CCA cell proliferation and migration through the GPER/PI3K/Akt/NF-κB pathway. This information can enhance public health awareness regarding ATZ contamination to prevent the relative risk of CCA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work report in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Opisthorchis viverrini (O. viverrini, Ov) infection and consumption of high-fat and high-fructose (HFF) diet exacerbate liver and kidney disease. Here, we investigated the effects of a combination of O. viverrini infection and HFF diet on kidney pathology via changes in the gut microbiome and host proteome in hamsters., Methodology/principal Findings: Twenty animals were divided into four groups; 1) fed a normal diet not infected with O. viverrini (normal group), 2) fed an HFF diet and not infected with O. viverrini (HFF), 3) fed a normal diet and infected with O. viverrini (Ov), and 4) fed an HFF diet and infected with O. viverrini (HFFOv). DNA was extracted from fecal samples and the V3-V4 region of the bacterial 16S rRNA gene sequenced on an Illumina MiSeq sequencing platform. In addition, LC/MS-MS analysis was done. Histopathological studies and biochemical assays were also conducted. The results indicated that the HFFOv group exhibited the most severe kidney injury, manifested as elevated KIM-1 expression and accumulation of fibrosis in kidney tissue. The microbiome of the HFFOv group was more diverse than in the HFF group: there were increased numbers of Ruminococcaceae, Lachnospiraceae, Desulfovibrionaceae and Akkermansiaceae, but fewer Eggerthellaceae. In total, 243 host proteins were identified across all groups. Analysis using STITCH predicted that host proteome changes may lead to leaking of the gut, allowing molecules such as soluble CD14 and p-cresol to pass through to promote kidney disease. In addition, differential expression of TGF-beta-activated kinase 1 and MAP3K7-binding protein 2 (Tab2, involving renal inflammation and injury) are predicted to be associated with kidney disease., Conclusions/significance: The combination of HFF diet and O. viverrini infection may promote kidney injury through alterations in the gut microbiome and host proteome. This knowledge may suggest an effective strategy to prevent kidney disease beyond the early stages., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Tunbenjasiri et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Microcystin-leucine arginine (MC-LR) is a cyanobacterial hepatotoxic toxin found in water sources worldwide, including in northeastern Thailand, where opisthorchiasis-associated cholangiocarcinoma (CCA) is most prevalent. MC-LR is a potential carcinogen; however, its involvement in liver fluke-associated CCA remains ambiguous. Here, we aimed to evaluate the effect of MC-LR on the progression of CCA via the Wnt/β-catenin pathway in vitro ., Methods: Cell division, migration, cell cycle transition, and MC-LR transporter expression were evaluated in vitro through MTT assay, wound healing assay, flow cytometry, and immunofluorescence staining, respectively. Following a 24-h treatment of cultured cells with 1, 10, 100, and 1,000 nM of MC-LR, the proliferative effect of MC-LR on the Wnt/β-catenin signaling pathway was investigated using immunoblotting and qRT-PCR analysis. Immunohistochemistry was used to determine β-catenin expression in CCA tissue compared to adjacent tissue., Results: Human immortalized cholangiocyte cells (MMNK-1) and a human cell line established from opisthorchiasis-associated CCA (KKU-213B) expressed the MC-LR transporter and internalized MC-LR. Exposure to 10 nM and 100 nM of MC-LR notably enhanced cells division and migration in both cell lines (P < 0.05) and markedly elevated the percentage of S phase cells (P < 0.05). MC-LR elevated PP2A expression by activating the Wnt/β-catenin signaling pathway and suppressing phosphatase activity. Inhibition of the β-catenin destruction complex genes ( Axin1 and APC ) led to the upregulation of β-catenin and its downstream target genes ( Cyclin D1 and c-Jun ). Inhibition of Wnt/β-catenin signaling by MSAB confirmed these results. Additionally, β-catenin was significantly expressed in cancerous tissue compared to adjacent areas (P < 0.001)., Conclusions: Our findings suggest that MC-LR promotes cell proliferation and progression of CCA through Wnt/β-catenin pathway. Further evaluation using in vivo experiments is needed to confirm this observation. This finding could promote health awareness regarding MC-LR intake and risk of CCA., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Background and Aim: Cholangiocarcinoma (CCA) is usually diagnosed at a late stage, leading to treatment failure. Cannabidiol (CBD), exhibits diverse anti-cancer effects in various cancers, offering avenues for improving CCA treatment. This study investigated the effects of CBD on human CCA cells and the underlying mechanisms in vitro and in vivo ., Experimental Procedure: The effects of CBD on three CCA cell lines (KKU-213B, KKU-100, KKU-055) were assessed using the SRB assay, clonogenic assay, cell cycle arrest, and 3D holotomography. Morphological changes were examined using transmission electron microscopy, while mitochondrial ROS levels and mitochondrial membrane potential were studied using MitoSOX, JC-1, and DCFH-DA. Cellular senescence induction was evaluated via SA-β-gal staining. Protein associatedwith autophagy and cellular senescence were analyzed using Western blot and/or immunofluorescent assays. A xenograft model demonstrated the anti-tumor activity of CBD and the induction of cellular senescence through immunohistochemistry targeting PCNA, β-gal, and p21., Results and Conclusion: CBD effectively inhibited CCA cell proliferation, suppressed colony formation and induced G0/G1 phase cell cycle arrest. Morphological examination revealed lipid droplets/vesicles in CCA cell lines. CBD induced autophagy by upregulating LC3BII, downregulating p62, and inhibiting the p -PI3K, p -AKT, and p -mTOR pathways. Additionally, CBD disrupted mitochondrial homeostasis by elevating ROS, reducing membrane potential, and induced cellular senescence by increasing the expression of p53 and p21. In-vitro results were confirmed by xenograft models. Overall, CBD suppresses proliferation and induces cell death, autophagy and senescence in CCA cells via the PI3K/AKT/mTOR pathway, which indicates a therapeutic option for CCA treatment., (© 2024 Center for Food and Biomolecules, National Taiwan University. Production and hosting by Elsevier Taiwan LLC.)