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 chol-angiocarcinoma cells (CCA) and the role of reactive oxygen generation in sensitizing the cells. METHODS: Four human cell lines with different NQO1 activity were used; the human CCA cell lines, KKU-100, KKU-OCA17, KKU-M214, and Chang liver cells. NQO1 activity and mRNA expression were determined. The cells were pretreated with dicoumarol at relevant concentrations before treatment with gemcitabine. Cytotoxicity was determined by staining with fluorescent dyes. Oxidant formation was examined by assay of cellular glu-tathione levels and reactive oxygen species production by using dihydrofluorescein diacetate. Measurement of mitochondrial transmembrane potential was performed by using JC-1 fluorescent probe. Western blotting analysis was performed to determine levels of survival related proteins. RESULTS: Dicoumarol markedly enhanced the cytotoxicity of gemcitabine in KKU-100 and KKU-OCA17, the high NQO1 activity and mRNA expressing cells, but not in the other cells with low NQO1 activity. Dicoumarol induced a marked decrease in cellular redox of gluta-thione in KKU-100 cells, in contrast to KKU-M214 cells. Dicoumarol at concentrations that inhibited NQO1 activity did not alter mitochondrial transmembrane potential and production of reactive oxygen species. Gemcitabine alone induced activation of NF-κB and Bcl-XL protein expression. However, gemcitabine and dicoumarol combination induced increased p53 and decreased Bcl-XL levels in KKU-100, but not in KKU-M214 cells. CONCLUSION: NQO1 may be important in sensitizing cells to anticancer drugs and inhibition of NQO1 may be a strategy for the treatment of CCA.

Rice bran hydrolysates induce immunomodulatory effects by suppression of chemotaxis, and modulation of cytokine release and cell-mediated cytotoxicity

Objective: To evaluate the immunomodulatory effects of rice bran hydrolysates on cultured immune cells and their underlying mechanism.Methods: Rice bran hydrolysates were prepared from pigmented rice(Oryza sativa L.) by hydrothermolysis and protease digestion. Rice bran hydrolysates were assayed for phenolic content and antioxidant activity. Cell proliferation of Jurkat, THP-1 and peripheral blood mononuclear cells(PBMC) was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Chemotaxis was evaluated by transwell chamber methods. Immunoadherence of THP-1 was performed on cultured human umbilical vein endothelial cells(HUVEC). Cytokine released from PBMC was measured by ELISA assay kits. Lymphocyte-mediated cytotoxicity was carried out on KKU-452 cells. Proteins associated with immunomodulation were analyzed by Western immunoblotting assay. Results: Rice bran hydrolysates were rich in phenolic compounds, such as ferulic acid, catechin, quercetin, and quercetin glycosides. Rice bran hydrolysates suppressed phytohemagglutinin(PHA)-stimulated proliferation of PBMC and Jurkat cells, chemotaxis of Jurkat and THP-1 cells, and immunoadherence of THP-1 on HUVEC cultured cells. The cellular mechanism of rice bran hydrolysates involved the activation of AMPK as well as suppression of m TOR, NF-κB and VCAM-1. Rice bran hydrolysates potentiated PBMC on the PHA-stimulated release of IL-2, TNF-α, and IL-4, and enhanced PHA-induced non-MHC-restricted cytotoxicity on KKU-452 cancer cells. Conclusions: The immunomodulatory effect of phytochemicals derived from rice bran hydrolysates suggests its therapeutic potential for further investigation.

Anti-tumor activity of rice bran hydrolysates on migration, invasion and angiogenesis

Objective:To investigate anti-tumor effect of rice bran hydrolysates(RBH)on proliferation,migration,invasion,and angiogenesis of cholangiocarcinoma(CCA)cells,and elucidate the underlying mechanisms.Methods:RBH was prepared from Tubtim Chumprae rice(Oryza sativa L.)by hydrothermolysis followed by protease digestion.Phenolic content in RBH was analyzed by high-performance liquid chromatography.Human CCA cells,KKU-156,KKU-452,and KKU-100,were used to study the effects of RBH on proliferation,migration,invasion,and adhesion by wound healing,Transwell chamber,and fibronectin cell adhesion assays.Angiogenesis was evaluated using human umbilical vein endothelial cells.Proteins associated with cancer progression were analyzed by immunobloting assays.Results:RBH contained carbohydrates,proteins,lipids,and various phenolic compounds and flavonoids.RBH did not inhibit CCA proliferation,but strongly suppressed migration,invasion,adhesion of CCA cells,and the formation of tube-like capillary structures of human umbilical vein endothelial cells.Moreover,RBH downregulated phosphorylation of FAK,PI3K,and Akt,suppressed NF-κB nuclear translocation,decreased the expression of ICAM-1,vimentin and vascular endothelium growth factor(VEGF),and increased the expression of E-cadherin.Conclusions:RBH suppresses CCA cell migration and invasion and decreases expression of proteins involved in cancer metastasis.RBH is a potential food supplement for cancer prevention.

Inflammatory cytokines suppress arylamine N-acetyltransferase 1 in cholangiocarcinoma cells

瞄准：为了在芳基胺上评估煽动性的 cytokines 的效果， N-acetyltransferase 1 (NAT1 ) 它是 phase-II 酶在在食物，药和环境发现的芳香、杂环的胺的简历转变包含了。方法：人的 cholangiocarcinoma KKU-100 房间与专业版的混合物被对待为 48 h 的煽动性的 cytokines (interferon-gamma， interleukin-1beta 和肿瘤坏死 factor-alpha ) ，和在 NAT1 活动的效果被高效液相色谱法估计，当 NAT1 表示被反向抄写的聚合酶链反应决定时。房间上的氧化压力被氮的氧化物，超级氧化物阴离子和谷胱甘肽(GSH ) 的形成检验层次。房间也与 S-nitroso-glutathione (GSNO ) 被对待，一个氮的氧化物施主，与煽动性的 cytokines 获得了看回答是否类似于那些。结果：Cytokines 压制了 NAT1 活动，减少没有影响 Km 的 Vmax。Cytokines 也在氮的氧化物生产并且在减少谷胱甘肽(GSH ) 和 GSH 二硫化物的氧化还原作用比率的正式就职上有重要影响。没有影响 GSH 比率，有为 2-48 h 的 GSNO 的处理减少了 NAT1 活动。而且，煽动性的 cytokines 和 GSNO 压制了 NAT1 mRNA 表示。结论：这些调查结果显示在 NAT1 的发炎和抑制之间的一个协会，它也许贡献调停化学药品的毒性和致癌作用。