Antitumor effects of candidone extracted from Derris indica (Lamk) Bennet in cholangiocarcinoma cells

Purpose: To investigate the antitumor effect of candidone extracted from Derris indica, against human cholangiocarcinoma (CCA) cells.Methods: Candidone was purified from the hexane extract of Derris indica fruit. CCA cell lines, KKUM156 and KKU-M213, were treated with candidone. Sulforhodamine B (SRB) assay and acridine orange/ethidium bromide (AO/EB) staining were used to investigate the effects of candidone on cell proliferation and induction of apoptosis, respectively. The effect on cell migration was assessed by wound healing assay. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was employed to assess the effects of candidone on the expression of genes that regulate proliferation and apoptosis.Results: Candidone exerted strong anticancer effects on CCA cells. The agent suppressed CCA cell proliferation and induced apoptotic cell death. RT-qPCR assay revealed that candidone significantly increased the expression of anti-proliferative and pro-apoptotic genes, including p21 and Bax, and decreased the expression of anti-apoptotic genes, including Bcl-2 and survivin. Moreover, candidone inhibited the migration of CCA cells induced by IGF-1.Conclusion: Candidone exhibits potent antitumor effect on CCA cells. These findings suggest that candidone is potentially suitable for the management of CCA and, therefore, warrants further investigation.Keywords: Candidone, Derris indica, Cholangiocarcinoma, Cytotoxicity, Apoptosi

Crucial Role of Heme Oxygenase-1 on the Sensitivity of Cholangiocarcinoma Cells to Chemotherapeutic Agents

Cancer cells acquire drug resistance via various mechanisms including enhanced cellular cytoprotective and antioxidant activities. Heme oxygenase-1 (HO-1) is a key enzyme exerting potent cytoprotection, cell proliferation and drug resistance. We aimed to investigate roles of HO-1 in human cholangiocarcinoma (CCA) cells for cytoprotection against chemotherapeutic agents. KKU-100 and KKU-M214 CCA cell lines with high and low HO-1 expression levels, respectively, were used to evaluate the sensitivity to chemotherapeutic agents, gemcitabine (Gem) and doxorubicin. Inhibition of HO-1 by zinc protoporphyrin IX (ZnPP) sensitized both cell types to the cytotoxicity of chemotherapeutic agents. HO-1 gene silencing by siRNA validated the cytoprotective effect of HO-1 on CCA cells against Gem. Induction of HO-1 protein expression by stannous chloride enhanced the cytoprotection and suppression of apoptosis caused by anticancer agents. The sensitizing effect of ZnPP was associated with increased ROS formation and loss of mitochondrial transmembrane potential, while Gem alone did not show any effects. A ROS scavenger, Tempol, abolished the sensitizing effect of ZnPP on Gem. Combination of ZnPP and Gem enhanced the release of cytochrome c and increased p21 levels. The results show that HO-1 played a critical role in cytoprotection in CCA cells against chemotherapeutic agents. Targeted inhibition of HO-1 may be a strategy to overcome drug resistance in chemotherapy of bile duct cancer

Dicoumarol enhances gemcitabine-induced cytotoxicity in high NQO1-expressing cholangiocarcinoma cells

AIM: To investigate whether dicoumarol, a potent inhibitor of NAD(P)H quinone oxidoreductase-1 (NQO1), potentiates gemcitabine to induce cytotoxicity in cholangiocarcinoma cells (CCA) and the role of reactive oxygen generation in sensitizing the cells

Therapeutic targeting of ARID1A and PI3K/AKT pathway alterations in cholangiocarcinoma

Background Genetic alterations in ARID1A were detected at a high frequency in cholangiocarcinoma (CCA). Growing evidence indicates that the loss of ARID1A expression leads to activation of the PI3K/AKT pathway and increasing sensitivity of ARID1A-deficient cells for treatment with the PI3K/AKT inhibitor. Therefore, we investigated the association between genetic alterations of ARID1A and the PI3K/AKT pathway and evaluated the effect of AKT inhibition on ARID1A-deficient CCA cells. Methods Alterations of ARID1A, PI3K/AKT pathway-related genes, clinicopathological data and overall survival of 795 CCA patients were retrieved from cBio Cancer Genomics Portal (cBioPortal) databases. The association between genetic alterations and clinical data were analyzed. The effect of the AKT inhibitor (MK-2206) on ARID1A-deficient CCA cell lines and stable ARID1A-knockdown cell lines was investigated. Cell viability, apoptosis, and expression of AKT signaling were analyzed using an MTT assay, flow cytometry, and Western blots, respectively. Results The analysis of a total of 795 CCA samples revealed that ARID1A alterations significantly co-occurred with mutations of EPHA2 (p < 0.001), PIK3CA (p = 0.047), and LAMA1 (p = 0.024). Among the EPHA2 mutant CCA tumors, 82% of EPHA2 mutant tumors co-occurred with ARID1A truncating mutations. CCA tumors with ARID1A and EPHA2 mutations correlated with better survival compared to tumors with ARID1A mutations alone. We detected that 30% of patients with PIK3CA driver missense mutations harbored ARID1A-truncated mutations and 60% of LAMA1-mutated CCA co-occurred with truncating mutations of ARID1A. Interestingly, ARID1A-deficient CCA cell lines and ARID1A-knockdown CCA cells led to increased sensitivity to treatment with MK-2206 compared to the control. Treatment with MK-2206 induced apoptosis in ARID1A-knockdown KKU-213A and HUCCT1 cell lines and decreased the expression of pAKTS473 and mTOR. Conclusion These findings suggest a dependency of ARID1A-deficient CCA tumors with the activation of the PI3K/AKT-pathway, and that they may be more vulnerable to selective AKT pathway inhibitors which can be used therapeutically

sj-pdf-1-ict-10.1177_15347354221124861 – Supplemental material for Anti-metastatic Potential of Natural Triterpenoid Cucurbitacin B Against Cholangiocarcinoma Cells by Targeting Src Protein

Supplemental material, sj-pdf-1-ict-10.1177_15347354221124861 for Anti-metastatic Potential of Natural Triterpenoid Cucurbitacin B Against Cholangiocarcinoma Cells by Targeting Src Protein by Putthaporn Kaewmeesri, Piman Pocasap, Veerapol Kukongviriyapan, Auemduan Prawan, Sarinya Kongpetch and Laddawan Senggunprai in Integrative Cancer Therapies</p

Alteration of expression of proteins related to cell proliferation and apoptosis.

<p>The Western blots of p21 and cytochrome c protein expression in KKU-100 cells after treatment with Gem (0.01 µM)+/−ZnPP (0.1 µM) for 24 h. The levels of p21 and cytochrome c were normalized with β-actin as a loading control. The bars are mean±SEM, each from three independent experiments. *<i>p</i><0.05 vs untreated controls.</p

Induction of HO-1 suppressed the cytotoxicity of Gem and Dox.

<p>(<b>A</b>) The time-course of HO-1 induction by SnCl₂ (10 µM) in KKU-100 and KKU-M214 cells. The cells were cultured for overnight and exposed to SnCl₂ for 3, 6 and 24 h before whole cell lysates were collected and HO-1 protein was determined by the Western blotting, using β-actin; as loading control. The HO-1 protein expression in KKU-100 and KKU-M214 cells is relative stable during the first 6 h of exposure to SnCl₂, whereas high HO-1 expression is evident during 6 to 24 h. The cytotoxicity of Gem (0.1 µM) and Dox (0.5 µM) with or without SnCl₂ (10 µM) was determined in both cell lines after incubation for 24 h. The cell viability (<b>B</b>), apoptotic and necrotic cells (<b>C</b>) were evaluated by fluorescent dye staining. Data represent mean±SEM, each from three separated experiments. <b>*</b>, <i>p</i><0.05 vs drug alone.</p

Pathogenesis of cholangiocarcinoma: From genetics to signalling pathways

10.1016/j.bpg.2015.02.002Best Practice and Research: Clinical Gastroenterology292233-24

Knockdown of HO-1 by siRNA sensitized KKU-100 cells to Gem.

<p>The mRNA (A) and protein (B) levels of HO-1expression in KKU-100 cells are shown. KKU-100 cells were transfected with siRNA against HO-1 for 24 h and total RNA was prepared and analyzed by reverse transcription-PCR. In similar experiments, cell lysates were collected and HO-1 was determined by the Western blotting in KKU-100 cells, using β-actin, as loading control. (<b>C</b>) The cytotoxicity and IC50 of Gem in knocked down KKU-100 cells was determined. After transfection for 24 h, KKU-100 cells were treated with varied concentrations of Gem (0.001, 0.01, 0.05 and 0.1 µM) for another 24 h. The cell viability was evaluated by fluorescent dye staining. Data represent mean±SEM, each from three separated experiments. <b>*</b>, <i>p</i><0.05 vs non target knocked down cells.</p