Inflammatory cytokines suppress arylamine N -acetyltransferase 1 in cholangiocarcinoma cells

Abstract

B e n j a p o r n B u r a n r a t , A u e m d u a n P r a w a n , V e e r a p o l Kukongviriyapan, Abstract AIM: To evaluate the effect of inflammatory cytokines on arylamine N -acetyltransferase 1 (NAT1), which is a phase-Ⅱ enzyme involved in the biotransformation of aromatic and heterocyclic amines found in food, drugs and the environment. METHODS:Human cholangiocarcinoma KKU-100 cells were treated with a mixture of proinflammatory cytokines (interferon-γ, interleukin-1β, and tumor necrosis factor-α) for 48 h, and the effect on NAT1 activity was assessed by high performance liquid chromatography, while NAT1 expression was determined by reverse-transcription polymerase chain reaction. The oxidative stress on the cells was examined by the formation of nitric oxide, superoxide anion and glutathione (GSH) levels. The cells were also treated with S -nitroso-glutathione (GSNO), a nitric oxide donor, to see if the responses were similar to those obtained with the inflammatory cytokines. RESULTS:Cyt okines suppressed NAT1 activity, reducing the Vmax without affecting the K m. Cytokines also had a significant impact on the induction of nitric oxide production and in reducing the redox ratios of glutathione (GSH) and GSH disulfide. Treatment with GSNO for 2-48 h reduced NAT1 activity without affecting the GSH ratio. Moreover, inflammatory cytokines and GSNO suppressed NAT1 mRNA expression. CONCLUSION:These findings indicate an association between inflammation and suppression of NAT1, which perhaps contributes to chemical-mediated toxicity and carcinogenesis.

Haem oxygenase 1 expression is associated with prognosis in cholangiocarcinoma patients and with drug sensitivity in xenografted mice

“…The three CCA cell lines, KKU‐100, M213 and M055, were seeded at 1.5 × 10 4 cells/well in six‐well plates and allowed to grow for 24 h. Total RNA was isolated and PCR products were generated using a previously described method . Primer sequences were as follows: HO‐1: forward, 5′‐CTG ACC CAT GAC ACC AAG GAC‐3′ and HO‐1 reverse: 5′‐AAA GCC CTA CAG CAA CTG TCG‐3′, GenBank accession number ; β‐actin: forward 5′‐AGT GTA GCC CAG GAT GCC CTT‐3′ and β‐actin: reverse, 5′‐GCC AAG GTC ATC CAT GAC AAC‐3′, Gen Bank accession number .…”

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

“…Cells were exposed to the transfection mixture for 6 h. At the end of incubation period, 1.5 mL of antibiotic-free Ham's F12 complete medium was added and the cells were cultured for an additional 18 h. The siGENOME non-targeting siRNA (D-001210-02-05: Dharmacon), as a negative control was introduced to the cells using the same protocol. Total RNA and protein were extracted from cells 24 h after transfection using previously described methods [20] , [21] . Efficiency of the transient transfection was determined by real-time polymerase chain reaction (PCR) using specific primers and Western blotting.…”

“…KKU-100 and KKU-M214 cells were seeded at the density of 1.5×10 5 cells/well in 6 well-plates and allowed to growth for 24 h. Total RNA was isolated using a previously described method [20] . Total RNA (1 µg) was then reverse transcribed to single-stranded cDNA by the ImProm-II™ reverse transcription system (Promega, WI, USA) at conditions of 42°C for 60 min.…”

“…To monitor the intracellular accumulation of ROS, the lucigenin-enhanced chemiluminescence method was used for detecting superoxide anion according to the previously described method [20] . Briefly, KKU-100 cells were cultured in 35-mm dishes overnight.…”

Isoniazid clearance is impaired among human immunodeficiency virus/tuberculosis patients with high levels of immune activation

“…In vitro studies with hepatocyte cell culture demonstrate that coculture of hepatocytes with proinflammatory cytokines downregulates the expression of cytochrome P450 isoenzymes, in particular CYP3A4 . Moreover, there has been a single report of the downregulation of NAT‐1 expression in human cholangiocarcinoma cells after treatment with proinflammatory cytokines . Based on our findings, additional work to understand NAT‐2 regulation, isoniazid PK and TB treatment outcomes are warranted.…”