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1Department of Nutrition and Food Science, University of Maryland, College Park, MD, 20742, USA
2College of Food Engineering and Nutrition Science, Shaanxi Normal University, Xi’an, 741609, P. R. China
3Department of Bioresource Sciences, Andong National University, Andong, 760749, Republic of Korea
Tolfenamic acid (TA) is a traditional non-steroid anti-inflammatory drug (NSAID) and has been broadly used for the treatment of migraines. Nuclear factor kappa B (NF-κB) is a sequence-specific transcription factor and plays a key role in the development and progression of inflammation and cancer. We performed the current study to investigate the underlying mechanisms by which TA suppresses inflammation focusing on NF-κB pathway in TNF-α stimulated human normal and cancer cell lines and lipopolysaccharide (LPS)-stimulated mouse macrophages. Different types of human cells (HCT116, HT-29 and HEK293) and mouse macrophages (RAW264.7) were pre-treated with different concentrations of TA and then exposed to inflammatory stimuli such as TNF-α and LPS. Transcriptional activity of NF-κB, IκB-α-degradation, p65 translocation and mitogen-activated protein kinase (MAPK) activations were measured using luciferase assay and Western blots. Pre-treatment of TA repressed TNF-α- or LPS-stimulated NF-κB transactivation in a dose-dependent manner. TA treatment reduced degradation of IκB-α and subsequent translocation of p65 into nucleus. TA significantly down-regulated the phosphorylation of c-Jun N-terminal kinase (JNK). However, TA had no effect on NF-κB signaling and JNK phosphorylation in HT-29 human colorectal cancer cells. TA possesses anti-inflammatory activities through suppression of JNK/NF-κB pathway in different types of cells.
Many non-steroid anti-inflammatory drugs (NSAIDs) including aspirin, indomethacin, and sulindac exert antipyretic, antirheumatoid, and anti-inflammatory as well as anticancer activities. The major anti-inflammatory efficacy of NSAIDs is partly attributed to the selective inhibitory effect on cyclooxygenase (COX) activity and thereby synthesis of prostaglandin (PG) which act as inflammatory mediators (Proudman and McMillan, 1991).
Tolfenamic acid (TA) is a widely used NSAID and structurally resembles other fenamates (Corell, 1994). TA showed anti-inflammatory activity through inhibition of leukotriene B4 (LTB4)-induced chemotaxis (Kankaanranta
Nuclear factor κB (NF-κB) signaling pathway is a primary target for the anti-inflammatory effects of many NSAIDs (Kopp and Ghosh, 1994; Yin
In the current study, we hence evaluated whether TA can influence NF-κB pathway in TNF-α stimulated cancer cell lines (HCT116, HT-29 and HEK293) and LPS-stimulated RAW264.7 cell line, and we further explore the underlying mechanism.
The human colorectal cancer cell line (HCT116, HT-29), the human embryonic kidney cell line (HEK 293) and the mouse macrophage cell line (RAW264.7) were purchased from American Type Culture Collection (Manassas, VA, USA). Cell culture media was obtained from Invitrogen (Carlsbad, CA, USA). TA was purchased from Cayman Chemical (Ann Arbor, MI, USA). Tumor necrosis factor-α (TNF-α) and lipopolysaccharide (LPS) were from Sigma Aldrich (St. Lous, MO, USA).
All cells were maintained in Dulbecco’s Modified Eagle medium (DMEM/F-12) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator at 37°C and 5% CO2.
The transient transfection was performed using PolyJet DNA reagent (SignaGen Laboratories, Ijamsville, MD, USA) according to the manufacture’s protocol. Briefly, after seeding at 2×105 cells/well in 12-well plates and overnight culture, cells were transiently transfected with a plasmid mixture containing 1 μg of
After the treatment as indicated in the figure legends, cells were harvested and lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate) containing protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO) and phosphatase inhibitor cocktail (Sigma Aldrich) by incubation on ice for 30 min and subsequent centrifugation at 14,000 g for 10 min at 4°C. The supernatant was collected and protein concentration was quantified by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA). Proteins were resolved on SDS-PAGE gels and transferred onto nitrocellulose membranes (Osmonics, Minnetonka, MN, USA). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with 0.05% Tween 20 (TBS-T) for 1 h at room temperature and then probed with the appropriate primary antibodies in 5% nonfat dry milk in TBS-T at 4°C overnight. After washing three times in TBS-T, the blots were further incubated with the correct secondary antibodies conjugated with horse radish-peroxidase for 1 h at room temperature, and finally chemiluminescence was examined with Pierce ECL Western blotting substrate (Thermo Scientific) and visualized by ChemiDoc MP Imaging system (Bio-Rad, Hercules, CA, USA).
The cytosolic and nuclear fractions were prepared using a nuclear extract kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s instruction.
Statistical analysis was performed with student’s t-test, and
Our previous study indicated that the treatment of TA increases transcriptional activity of NF-κB in human colorectal cancer cells (Jeong
It is generally accepted that the phosphorylation and degradation of IκB-α is crucial for the nuclear translocation and subsequent activation of NF-κB. Thus, we performed Western blotting to determine if TA affects TNF-α- or LPS-stimulated IκB-α degradation in different cells. Pretreatment with TA dose-dependently ameliorated TNF-α-induced degradation of IκB-α in HCT116 (Fig. 2A) and HEK293 (Fig. 2B) cells, and LPS-induced RAW246.7 (Fig. 2C) cells, respectively. It is noted that, in TNF-α induced HT-29 cells, TA pretreatment showed a biphasic effect which blocked the IκB-α degradation at low doses (5 and 10 μM) but did not change at high doses (30 and 50 μM) (Fig. 2D).
Next, we further examined whether TA can abrogate inflammatory stimuli-mediated NF-κB translocation, which is a consequence of IκB-α degradation. As shown in Fig. 3, TA pretreatment decreased the levels of NF-κB (p65) in nucleus in a dose-dependent manner, whereas TNF-α or LPS only treatment dramatically increased nuclear NF-κB in HCT116, HEK29 and RAW264.7 cells, respectively. However, NF-κB level remained no significant change in HT-29 cells co-treated with TA and TNF-α.
Mitogen activated protein-kinases (MAPKs) including ERK-1/2, c-Jun and p38 are implicated in varied signaling cascades wherein various extracellular stimuli induce infiammation. And NF-κB is one of the well-established downstream targets of the MAPK signaling pathway. Therefore, the inhibitory effects of TA on TNFα- or LPS-stimulated expression of phospho-MAPKs were studied. As shown in Fig. 4. TNF-α (HCT116, HEK293 and HT-29 cells) or LPS treatment (RAW264.7) dramatically stimulated phosphorylation of all MAPKs. Pretreatment of TA significantly suppressed the expression of phospho-JNK in HCT116 (Fig. 4A), HEK293 (Fig. 4B), and RAW264.7 cells (Fig. 4C). However, TA did not influence TNF-α-induced JNK phosphorylation in HT-29 cells (Fig. 4D). Overall, phosphorylation of ERK and p38 was not significantly affected by the treatment of TA although decrease of ERK phosphorylation and slight increase of p38 phosphorylation was only observed in 50 μM TA-treated HEK293 cells, HCT116 and HT-29, respectively. Taken together, this result suggests that TA inhibits the activation of NF-κB through phosphorylation of JNK.
TA activates NF-κB signaling in the absence of inflammatory stimuli (Jeong
Next, we tested phosphorylation of JNK. TA treatment did not change phosphorylation of JNK whereas in the presence of TNF-α, TA suppressed phosphor-JNK (Fig. 5B). We also tested phosphorylation of ERK. In the absence of TNF-α, TA induced phosphorylation of ERK, which is consistent with our previous data (Lee
During the past a few decades, several NSAIDs have held considerable interest due to their anti-inflammatory and cancer preventive properties. The first anti-cancer activity of TA has been studied by Dr. Safe’s group in Texas A&M University (Abdelrahim
Interestingly, in the present study, we found that TA blocked the TNFα- or LPS-induced NF-κB activation in several human cells and mouse macrophages. NF-κB activation is mainly regulated through the inhibitory IκB proteins, and the stimulus-induced degradation of IκB lead to the release and subsequent activation of NF-κB. NSAIDs such as aspirin and sulindac have been reported to inhibit NF-κB by inhibiting IκB-α phosphorylation (Kopp and Ghosh, 1994). Flufenamic acid which chemical structure is similar with TA, also suppressed the TNFα- or LPS-induced NF-κB activation (Paik
Emerging evidence suggest that mitogen-activated protein kinases (MAPKs) participate in the regulation of NF-κB activation. Our early observation showed that the TA-induced ROS generation results in ERK activation and DNA damage and thereby NF-κB and ATF3 activation, and eventually apoptosis in human colorectal cancer cells (Jeong
One of interesting findings is that TA effects on NF-κB signaling are determined by inflammatory status. For example, TA activates NF-κB signaling in the absence of inflammatory stimuli, whereas it suppressed NF-κB signaling in the presence of inflammatory stimuli. Dual effects of NSAIDs on NF-κB pathway were also observed in other studies. Aspirin activates NF-κB transcriptional activity, resulting in induction of apoptosis in human colorectal cancer cells (Stark
Another interesting finding is that anti-NF-κB signaling activity by TA is not observed in HT-29 cells. However, other NSAIDS such as aspirin suppress inflammation in HT-29 cells (Bergman
In summary, the data presented here shows that in absence of inflammatory stimuli, TA induces p65 nuclear accumulation and activates NF-κB transcriptional activity and apoptosis. However, in the presence of inflammatory stimulus, TA prevented inflammatory cytokine-induced NF-κB transcriptional activity. Collectively, our result shed more lights on the complex action mechanism of TA that the anti-inflammatory effects of TA are partially mediated through the blockage of JNK/NF- κB pathway.
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