Cholangiocarcinoma (CCA) is highly resistant to most of the known chemotherapeutic treatments. NAD(P)H-quinone oxidoreductase 1 (NQO1) is an antioxidant/detoxifying enzyme recently recognized as an important contributor to chemoresistance in some human cancers. However, the contribution of NQO1 to chemotherapy resistance in CCA is unknown.

Luteolin Arrests Cell Cycling, Induces Apoptosis and Inhibits the JAK/STAT3 Pathway in Human Cholangiocarcinoma Cells Luteolin;cholangiocarcinoma;cell cycle;apoptosis;JAK/STAT3;migration; Cholangiocarcinoma (CCA) is one of the aggressive cancers with a very poor prognosis. Several efforts have been made to identify and develop new agents for prevention and treatment of this deadly disease. In the present study, we examined the anticancer effect of luteolin on human CCA, KKU-M156 cells. Sulforhodamine B assays showed that luteolin had potent cytotoxicity on CCA cells with IC50 values of $10.5{\pm}5.0$ and $8.7{\pm}3.5{\mu}M$ at 24 and 48 h, respectively. Treatment with luteolin also caused a concentration-dependent decline in colony forming ability. Consistent with growth inhibitory effects, luteolin arrested cell cycle progression at the G2/M phase in a dose-dependent manner as assessed by flow cytometry analysis. Protein expression of cyclin A and Cdc25A was down-regulated after luteolin treatment, supporting the arrest of cells at the G2/M boundary. Besides evident G2/M arrest, luteolin induced apoptosis of KKU-M156 cells, demonstrated by a distinct sub-G1 apoptotic peak and fluorescent dye staining. A decrease in the level of anti-apoptotic Bcl-2 protein was implicated in luteolin-induced apoptosis. We further investigated the effect of luteolin on JAK/STAT3, which is an important pathway involved in the development of CCA. The results showed that interleukin-6 (IL-6)-induced JAK/STAT3 activation in KKU-M156 cells was suppressed by treatment with luteolin. Treatment with a specific JAK inhibitor, AG490, and luteolin diminished IL-6-stimulated CCA cell migration as assessed by wound healing assay. These data revealed anticancer activity of luteolin against CCA so the agent might have potential for CCA prevention and therapy.

Objectives: To investigate the apoptosis-inducing effect and underlying mechanisms of luteolin in cholangiocarcinoma (CCA) cells. Methods: Cell viability was determined by sulphorhodamine B. Apoptosis was detected using acridine orange/ethidium bromide dye staining and annexin V/PI staining followed by flow cytometry. The effect of luteolin on the oxidative status of CCA cells was evaluated by measuring intracellular reactive oxygen species (ROS) levels using the dihydroethidium method and quantifying glutathione levels. The mitochondria transmembrane potential (ΔΨm) was examined through JC-1 staining. The protein levels were determined by Western blot. Caspase activity was determined using specific fluorogenic substrates. Key findings: Luteolin decreased KKU-100 CCA cells’ viability by induction of apoptosis. Luteolin treatment increased ROS production and decreased glutathione levels. These changes were associated with the decrease of Nrf2, γ-glutamylcysteine ligase and heme oxygenase-1 proteins. Moreover, luteolin induced mitochondrial depolarization, which was accompanied by the release of cytochrome c and a decrease of Bcl-2 and Bcl-XL proteins. Pretreatment with antioxidants, 4-hydroxy-TEMPO and N-acetyl-L-cysteine significantly prevented luteolin-induced CCA cell death and loss of ΔΨm. In addition, luteolin induced the activation of caspase-9 and caspase-3. Conclusions: Luteolin exerts its pro-apoptotic action partly through generating intracellular ROS that then contributes to the activation of mitochondria-mediated apoptotic cell death.

Phenethyl isothiocyanate (PEITC) is a cancer chemopreventive agent from cruciferous vegetables. Cholangiocarcinoma (CCA) is a chemo-resistant cancer with very poor prognosis. We evaluated the effects of PEITC on induction of apoptotic cell death in relation to cellular glutathione (GSH) and mitochondrial function of a CCA cell line, KKU-M214.

Owing to the crucial role of Src in cancer metastasis, interruption of Src and its signaling has been considered a promising strategy for cancer metastasis treatment. Cucurbitacin B, a dietary triterpenoid, has been shown to possess anti-proliferative and apoptosis-inducing activities in cholangiocarcinoma (CCA) cells via suppressing the activation of FAK which is a main downstream Src effector. We hypothesized that cucurbitacin B might act as a Src suppressant which conferring anti-metastasis effect against CCA cells. To investigate this, the role of Src in regulating metastasis behavior of CCA cells and the effect of cucurbitacin B on Src-mediated metastatic phenotype of these cells were determined. The results showed that activation of Src significantly enhanced the migratory and invasive abilities of CCA cells. Molecular analysis revealed that Src-facilitated metastasis behavior of CCA cells occurred by modifying expression of a wide range of metastasis-related genes in the cells. Consistent with gene expression results, activation of Src significantly induced the protein expression of 2 important metastasis-associated molecules, MMP-9 and VEGF. Cucurbitacin B markedly suppressed activation of Src and its key effector, FAK. As a consequence, the alteration of expression profiles of metastasis-associated genes induced by Src activator in CCA cells was diminished by cucurbitacin B treatment. The compound also down-regulated Src-induced expression of MMP-9 and VEGF proteins in the cells. Moreover, molecular docking analysis revealed that cucurbitacin B could interact with Src kinase domain and possibly restrain the kinase from being activated by hindering the ATP binding. In conclusion, cucurbitacin B exhibited anti-metastatic property in CCA cells via negatively influencing Src and Src-related oncogenic signaling. This compound may therefore be a potential therapeutic drug for further development as an anti-Src agent for treatment of metastatic CCA.